Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription

ABSTRACT

The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/385,360 filed Apr. 16, 2019, which is a continuation of U.S. patentapplication Ser. No. 13/842,859 filed Mar. 15, 2013, now U.S. Pat. No.10,266,850 issued Apr. 23, 2019, which claims the benefit of U.S.Provisional Patent Application Nos. 61/652,086 filed May 25, 2012,61/716,256 filed Oct. 19, 2012, 61/757,640 filed Jan. 28, 2013, and61/765,576, filed Feb. 15, 2013, each of which applications isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. GM081879awarded by the National Institutes of Health. The government has certainrights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“BERK-187-SeqList_ST25.txt” created on Mar. 14, 2013 and having a sizeof 7645 KB. The contents of the text file are incorporated by referenceherein in their entirety.

BACKGROUND

About 60% of bacteria and 90% of archaea possess CRISPR (clusteredregularly interspaced short palindromic repeats)/CRISPR-associated (Cas)system systems to confer resistance to foreign DNA elements. Type IICRISPR system from Streptococcus pyogenes involves only a single geneencoding the Cas9 protein and two RNAs—a mature CRISPR RNA (crRNA) and apartially complementary trans-acting RNA (tracrRNA)—which are necessaryand sufficient for RNA-guided silencing of foreign DNAs.

In recent years, engineered nuclease enzymes designed to target specificDNA sequences have attracted considerable attention as powerful toolsfor the genetic manipulation of cells and whole organisms, allowingtargeted gene deletion, replacement and repair, as well as the insertionof exogenous sequences (transgenes) into the genome. Two majortechnologies for engineering site-specific DNA nucleases have emerged,both of which are based on the construction of chimeric endonucleaseenzymes in which a sequence non-specific DNA endonuclease domain isfused to an engineered DNA binding domain. However, targeting each newgenomic locus requires the design of a novel nuclease enzyme, makingthese approaches both time consuming and costly. In addition, bothtechnologies suffer from limited precision, which can lead tounpredictable off-target effects.

The systematic interrogation of genomes and genetic reprogramming ofcells involves targeting sets of genes for expression or repression.Currently the most common approach for targeting arbitrary genes forregulation is to use RNA interference (RNAi). This approach haslimitations. For example, RNAi can exhibit significant off-targeteffects and toxicity.

There is need in the field for a technology that allows precisetargeting of nuclease activity (or other protein activities) to distinctlocations within a target DNA in a manner that does not require thedesign of a new protein for each new target sequence. In addition, thereis a need in the art for methods of controlling gene expression withminimal off-target effects.

SUMMARY

The present disclosure provides a DNA-targeting RNA that comprises atargeting sequence and, together with a modifying polypeptide, providesfor site-specific modification of a target DNA and/or a polypeptideassociated with the target DNA. The present disclosure further providessite-specific modifying polypeptides. The present disclosure furtherprovides methods of site-specific modification of a target DNA and/or apolypeptide associated with the target DNA The present disclosureprovides methods of modulating transcription of a target nucleic acid ina target cell, generally involving contacting the target nucleic acidwith an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA.Kits and compositions for carrying out the methods are also provided.The present disclosure provides genetically modified cells that produceCas9; and Cas9 transgenic non-human multicellular organisms.

FEATURES

Features of the present disclosure include a DNA-targeting RNAcomprising: (i) a first segment comprising a nucleotide sequence that iscomplementary to a sequence in a target DNA; and (ii) a second segmentthat interacts with a site-directed modifying polypeptide. In somecases, the first segment comprises 8 nucleotides that have 100%complementarity to a sequence in the target DNA. In some cases, thesecond segment comprises a nucleotide sequence with at least 60%identity over a stretch of at least 8 contiguous nucleotides to any oneof the nucleotide sequences set forth in SEQ ID NOs:431-682 (e.g.,431-562). In some cases, the second segment comprises a nucleotidesequence with at least 60% identity over a stretch of at least 8contiguous nucleotides to any one of the nucleotide sequences set forthin SEQ ID NOs:563-682. In some cases, the site-directed modifyingpolypeptide comprises an amino acid sequence having at least about 75%amino acid sequence identity to amino acids 7-166 or 731-1003 of theCas9/Csn1 amino acid sequence depicted in FIG. 3A and FIG. 3B, or to thecorresponding portions in any of the amino acid sequences set forth asSEQ ID NOs:1-256 and 795-1346.

Features of the present disclosure include a DNA polynucleotidecomprising a nucleotide sequence that encodes the DNA-targeting RNA. Insome cases, a recombinant expression vector comprises the DNApolynucleotide. In some cases, the nucleotide sequence encoding theDNA-targeting RNA is operably linked to a promoter. In some cases, thepromoter is an inducible promoter. In some cases, the nucleotidesequence encoding the DNA-targeting RNA further comprises a multiplecloning site. Features of the present disclosure include an in vitrogenetically modified host cell comprising the DNA polynucleotide.

Features of the present disclosure include a recombinant expressionvector comprising: (i) a nucleotide sequence encoding a DNA-targetingRNA, wherein the DNA-targeting RNA comprises: (a) a first segmentcomprising a nucleotide sequence that is complementary to a sequence ina target DNA; and (b) a second segment that interacts with asite-directed modifying polypeptide; and (ii) a nucleotide sequenceencoding the site-directed modifying polypeptide comprising: (a) anRNA-binding portion that interacts with the DNA-targeting RNA; and (b)an activity portion that exhibits site-directed enzymatic activity,wherein the site of enzymatic activity is determined by theDNA-targeting RNA.

Features of the present disclosure include a recombinant expressionvector comprising: (i) a nucleotide sequence encoding a DNA-targetingRNA, wherein the DNA-targeting RNA comprises: (a) a first segmentcomprising a nucleotide sequence that is complementary to a sequence ina target DNA; and (b) a second segment that interacts with asite-directed modifying polypeptide; and (ii) a nucleotide sequenceencoding the site-directed modifying polypeptide, where thesite-directed modifying polypeptide comprises: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that modulates transcription within the target DNA, wherein thesite of modulated transcription within the target DNA is determined bythe DNA-targeting RNA.

Features of the present disclosure include a variant site-directedmodifying polypeptide comprising: (i) an RNA-binding portion thatinteracts with a DNA-targeting RNA, wherein the DNA-targeting RNAcomprises a nucleotide sequence that is complementary to a sequence in atarget DNA; and (ii) an activity portion that exhibits reducedsite-directed enzymatic activity, wherein the site of enzymatic activityis determined by the DNA-targeting RNA. In some cases, the variantsite-directed modifying polypeptide comprises an H840A mutation of theS. pyogenes sequence SEQ ID NO:8 or the corresponding mutation in any ofthe amino acid sequences set forth as SEQ ID NOs:1-256 and 795-1346. Insome cases, the variant site-directed modifying polypeptide comprises aD10A mutation of the S. pyogenes sequence SEQ ID NO:8 or thecorresponding mutation in any of the amino acid sequences set forth asSEQ ID NOs:1-256 and 795-1346. In some cases, the variant site-directedmodifying polypeptide comprises both (i) a D10A mutation of the S.pyogenes sequence SEQ ID NO:8 or the corresponding mutation in any ofthe amino acid sequences set forth as SEQ ID NOs:1-256 and 795-1346; and(ii) an H840A mutation of the S. pyogenes sequence SEQ ID NO:8 or thecorresponding mutation in any of the amino acid sequences set forth asSEQ ID NOs:1-256 and 795-1346.

Features of the present disclosure include a chimeric site-directedmodifying polypeptide comprising: (i) an RNA-binding portion thatinteracts with a DNA-targeting RNA, wherein the DNA-targeting RNAcomprises a nucleotide sequence that is complementary to a sequence in atarget DNA; and (ii) an activity portion that exhibits site-directedenzymatic activity, wherein the site of enzymatic activity is determinedby the DNA-targeting RNA. In some cases, the chimeric site-directedmodifying polypeptide of comprises an amino acid sequence having atleast about 75% amino acid sequence identity to amino acids 7-166 or731-1003 of the Cas9/Csn1 amino acid sequence depicted in FIG. 3A andFIG. 3B, or to the corresponding portions in any of the amino acidsequences set forth as SEQ ID NOs:1-256 and 795-1346. In some cases, theDNA-targeting RNA further comprises a nucleotide sequence with at least60% identity over a stretch of at least 8 contiguous nucleotides to anyone of the nucleotide sequences set forth in SEQ ID NOs:431-682 (e.g.,SEQ ID NOs:563-682). In some cases, the DNA-targeting RNA furthercomprises a nucleotide sequence with at least 60% identity over astretch of at least 8 contiguous nucleotides to any one of thenucleotide sequences set forth in SEQ ID NOs:431-562. In some cases, theenzymatic activity of the chimeric site-directed modifying polypeptidemodifies the target DNA. In some cases, the enzymatic activity of thechimeric site-directed modifying polypeptide is nuclease activity,methyltransferase activity, demethylase activity, DNA repair activity,DNA damage activity, deamination activity, dismutase activity,alkylation activity, depurination activity, oxidation activity,pyrimidine dimer forming activity, integrase activity, transposaseactivity, recombinase activity, polymerase activity, ligase activity,helicase activity, photolyase activity or glycosylase activity. In somecases, the enzymatic activity of the chimeric site-directed modifyingpolypeptide is nuclease activity. In some cases, the nuclease activityintroduces a double strand break in the target DNA. In some cases, theenzymatic activity of the chimeric site-directed modifying polypeptidemodifies a target polypeptide associated with the target DNA. In somecases, the enzymatic activity of the chimeric site-directed modifyingpolypeptide is methyltransferase activity, demethylase activity,acetyltransferase activity, deacetylase activity, kinase activity,phosphatase activity, ubiquitin ligase activity, deubiquitinatingactivity, adenylation activity, deadenylation activity, SUMOylatingactivity, deSUMOylating activity, ribosylation activity, deribosylationactivity, myristoylation activity or demyristoylation activity.

Features of the present disclosure include a polynucleotide comprising anucleotide sequence encoding a chimeric site-directed modifyingpolypeptide. In some cases, the polynucleotide is an RNA polynucleotide.In some cases, the polynucleotide is a DNA polynucleotide. Features ofthe present disclosure include a recombinant expression vectorcomprising the polynucleotide. In some cases, the polynucleotide isoperably linked to a promoter. In some cases, the promoter is aninducible promoter. Features of the present disclosure include an invitro genetically modified host cell comprising the polynucleotide.

Features of the present disclosure include a chimeric site-directedmodifying polypeptide comprising: (i) an RNA-binding portion thatinteracts with a DNA-targeting RNA, wherein the DNA-targeting RNAcomprises a nucleotide sequence that is complementary to a sequence in atarget DNA; and (ii) an activity portion that modulates transcriptionwithin the target DNA, wherein the site of modulated transcriptionwithin the target DNA is determined by the DNA-targeting RNA. In somecases, the activity portion increases transcription within the targetDNA. In some cases, the activity portion decreases transcription withinthe target DNA.

Features of the present disclosure include a genetically modified cellcomprising a recombinant site-directed modifying polypeptide comprisingan RNA-binding portion that interacts with a DNA-targeting RNA; and anactivity portion that exhibits site-directed enzymatic activity, whereinthe site of enzymatic activity is determined by the DNA-targeting RNA.In some cases, the site-directed modifying polypeptide comprises anamino acid sequence having at least about 75% amino acid sequenceidentity to amino acids 7-166 or 731-1003 of the Cas9/Csn1 amino acidsequence depicted in FIG. 3A and FIG. 3B, or to the correspondingportions in any of the amino acid sequences set forth as SEQ IDNOs:1-256 and 795-1346. In some cases, the cell is selected from thegroup consisting of: an archaeal cell, a bacterial cell, a eukaryoticcell, a eukaryotic single-cell organism, a somatic cell, a germ cell, astem cell, a plant cell, an algal cell, an animal cell, in invertebratecell, a vertebrate cell, a fish cell, a frog cell, a bird cell, amammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, arodent cell, a rat cell, a mouse cell, a non-human primate cell, and ahuman cell.

Features of the present disclosure include a transgenic non-humanorganism whose genome comprises a transgene comprising a nucleotidesequence encoding a recombinant site-directed modifying polypeptidecomprising: (i) an RNA-binding portion that interacts with aDNA-targeting RNA; and (ii) an activity portion that exhibitssite-directed enzymatic activity, wherein the site of enzymatic activityis determined by the DNA-targeting RNA. In some cases, the site-directedmodifying polypeptide comprises an amino acid sequence having at leastabout 75% amino acid sequence identity to amino acids 7-166 or 731-1003of the Cas9/Csn1 amino acid sequence depicted in FIG. 3A and FIG. 3B, orto the corresponding portions in any of the amino acid sequences setforth as SEQ ID NOs:1-256 and 795-1346. In some cases, the organism isselected from the group consisting of: an archaea, a bacterium, aeukaryotic single-cell organism, an algae, a plant, an animal, aninvertebrate, a fly, a worm, a cnidarian, a vertebrate, a fish, a frog,a bird, a mammal, an ungulate, a rodent, a rat, a mouse, and a non-humanprimate.

Features of the present disclosure include a composition comprising: (i)a DNA-targeting RNA, or a DNA polynucleotide encoding the same, theDNA-targeting RNA comprising: (a) a first segment comprising anucleotide sequence that is complementary to a sequence in a target DNA;and (b) a second segment that interacts with a site-directed modifyingpolypeptide; and (ii) the site-directed modifying polypeptide, or apolynucleotide encoding the same, the site-directed modifyingpolypeptide comprising: (a) an RNA-binding portion that interacts withthe DNA-targeting RNA; and (b) an activity portion that exhibitssite-directed enzymatic activity, wherein the site of enzymatic activityis determined by the DNA-targeting RNA. In some cases, the first segmentof the DNA-targeting RNA comprises 8 nucleotides that have at least 100%complementarity to a sequence in the target DNA. In some cases, thesecond segment of the DNA-targeting RNA comprises a nucleotide sequencewith at least 60% identity over a stretch of at least 8 contiguousnucleotides to any one of the nucleotide sequences set forth in SEQ IDNOs:431-682 (e.g., SEQ ID NOs:563-682). In some cases, the secondsegment of the DNA-targeting RNA comprises a nucleotide sequence with atleast 60% identity over a stretch of at least 8 contiguous nucleotidesto any one of the nucleotide sequences set forth in SEQ ID NOs:431-562.In some cases, the site-directed modifying polypeptide comprises anamino acid sequence having at least about 75% amino acid sequenceidentity to amino acids 7-166 or 731-1003 of the Cas9/Csn1 amino acidsequence depicted in FIG. 3A and FIG. 3B, or to the correspondingportions in any of the amino acid sequences set forth as SEQ IDNOs:1-256 and 795-1346. In some cases, the enzymatic activity modifiesthe target DNA. In some cases, the enzymatic activity is nucleaseactivity, methyltransferase activity, demethylase activity, DNA repairactivity, DNA damage activity, deamination activity, dismutase activity,alkylation activity, depurination activity, oxidation activity,pyrimidine dimer forming activity, integrase activity, transposaseactivity, recombinase activity, polymerase activity, ligase activity,helicase activity, photolyase activity or glycosylase activity. In somecases, the enzymatic activity is nuclease activity. In some cases, thenuclease activity introduces a double strand break in the target DNA. Insome cases, the enzymatic activity modifies a target polypeptideassociated with the target DNA. In some cases, the enzymatic activity ismethyltransferase activity, demethylase activity, acetyltransferaseactivity, deacetylase activity, kinase activity, phosphatase activity,ubiquitin ligase activity, deubiquitinating activity, adenylationactivity, deadenylation activity, SUMOylating activity, deSUMOylatingactivity, ribosylation activity, deribosylation activity, myristoylationactivity or demyristoylation activity. In some cases, the targetpolypeptide is a histone and the enzymatic activity is methyltransferaseactivity, demethylase activity, acetyltransferase activity, deacetylaseactivity, kinase activity, phosphatase activity, ubiquitin ligaseactivity or deubiquitinating activity. In some cases, the DNA-targetingRNA is a double-molecule DNA-targeting RNA and the composition comprisesboth a targeter-RNA and an activator-RNA, the duplex-forming segments ofwhich are complementary and hybridize to form the second segment of theDNA-targeting RNA. In some cases, the duplex-forming segment of theactivator-RNA comprises a nucleotide sequence with at least 60% identityover a stretch of at least 8 contiguous nucleotides to any one of thenucleotide sequences set forth in SEQ ID NO:SEQ ID NOs:431-682.

Features of the present disclosure include a composition comprising: (i)a DNA-targeting RNA of the present disclosure, or a DNA polynucleotideencoding the same; and (ii) a buffer for stabilizing nucleic acids.Features of the present disclosure include a composition comprising: (i)a site-directed modifying polypeptide of the present disclosure, or apolynucleotide encoding the same; and (ii) a buffer for stabilizingnucleic acids and/or proteins. Features of the present disclosureinclude a composition comprising: (i) a DNA-targeting RNA, or a DNApolynucleotide encoding the same, the DNA-targeting RNA comprising: (a)a first segment comprising a nucleotide sequence that is complementaryto a sequence in a target DNA; and (b) a second segment that interactswith a site-directed modifying polypeptide; and (ii) the site-directedmodifying polypeptide, or a polynucleotide encoding the same, thesite-directed modifying polypeptide comprising: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that modulates transcription within the target DNA, wherein thesite of modulated transcription within the target DNA is determined bythe DNA-targeting RNA. In some cases, the activity portion increasestranscription within the target DNA. In some cases, the activity portiondecreases transcription within the target DNA. Features of the presentdisclosure include a composition comprising: (i) a site-directedmodifying polypeptide, or a polynucleotide encoding the same; and (ii) abuffer for stabilizing nucleic acids and/or proteins.

Features of the present disclosure include a method of site-specificmodification of a target DNA, the method comprising: contacting thetarget DNA with: (i) a DNA-targeting RNA, or a DNA polynucleotideencoding the same, wherein the DNA-targeting RNA comprises: (a) a firstsegment comprising a nucleotide sequence that is complementary to asequence in the target DNA; and (b) a second segment that interacts witha site-directed modifying polypeptide; and (ii) a site-directedmodifying polypeptide, or a polynucleotide encoding the same, whereinthe site-directed modifying polypeptide comprises: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that exhibits site-directed enzymatic activity. In some cases,the target DNA is extrachromosomal. In some cases, the target DNAcomprises a PAM sequence of the complementary strand that is 5′-CCY-3′,wherein Y is any DNA nucleotide and Y is immediately 5′ of the targetsequence of the complementary strand of the target DNA. In some cases,the target DNA is part of a chromosome in vitro. In some cases, thetarget DNA is part of a chromosome in vivo. In some cases, the targetDNA is part of a chromosome in a cell. In some cases, the cell isselected from the group consisting of: an archaeal cell, a bacterialcell, a eukaryotic cell, a eukaryotic single-cell organism, a somaticcell, a germ cell, a stem cell, a plant cell, an algal cell, an animalcell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell,a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, asheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primatecell, and a human cell. In some cases, the DNA-targeting RNA comprises anucleotide sequence with at least 60% identity over a stretch of atleast 8 contiguous nucleotides to any one of the nucleotide sequencesset forth in SEQ ID NOs:431-682 (e.g., SEQ ID NOs:563-682). In somecases, the DNA-targeting RNA comprises a nucleotide sequence with atleast 60% identity over a stretch of at least 8 contiguous nucleotidesto any one of the nucleotide sequences set forth SEQ ID NOs:431-562. Insome cases, the DNA-modifying polypeptide comprises an amino acidsequence having at least about 75% amino acid sequence identity to aminoacids 7-166 or 731-1003 of the Cas9/Csn1 amino acid sequence depicted inFIG. 3A and FIG. 3B, or to the corresponding portions in any of theamino acid sequences set forth as SEQ ID NOs:1-256 and 795-1346. In somecases, the enzymatic activity modifies the target DNA. In some cases,the enzymatic activity is nuclease activity, methyltransferase activity,demethylase activity, DNA repair activity, DNA damage activity,deamination activity, dismutase activity, alkylation activity,depurination activity, oxidation activity, pyrimidine dimer formingactivity, integrase activity, transposase activity, recombinaseactivity, polymerase activity, ligase activity, helicase activity,photolyase activity or glycosylase activity. In some cases, theDNA-modifying enzymatic activity is nuclease activity. In some cases,the nuclease activity introduces a double strand break in the targetDNA. In some cases, the contacting occurs under conditions that arepermissive for nonhomologous end joining or homology-directed repair. Insome cases, the method further comprises contacting the target DNA witha donor polynucleotide, wherein the donor polynucleotide, a portion ofthe donor polynucleotide, a copy of the donor polynucleotide, or aportion of a copy of the donor polynucleotide integrates into the targetDNA. In some cases, the method does not comprise contacting the cellwith a donor polynucleotide, wherein the target DNA is modified suchthat nucleotides within the target DNA are deleted. In some cases, theenzymatic activity modifies a target polypeptide associated with thetarget DNA. In some cases, the enzymatic activity is methyltransferaseactivity, demethylase activity, acetyltransferase activity, deacetylaseactivity, kinase activity, phosphatase activity, ubiquitin ligaseactivity, deubiquitinating activity, adenylation activity, deadenylationactivity, SUMOylating activity, deSUMOylating activity, ribosylationactivity, deribosylation activity, myristoylation activity ordemyristoylation activity. In some cases, the target polypeptide is ahistone and the enzymatic activity is methyltransferase activity,demethylase activity, acetyltransferase activity, deacetylase activity,kinase activity, phosphatase activity, ubiquitin ligase activity ordeubiquitinating activity. In some cases, the complex further comprisesan activator-RNA. In some cases, the activator-RNA comprises anucleotide sequence with at least 60% identity over a stretch of atleast 8 contiguous nucleotides to any one of the nucleotide sequencesset forth in SEQ ID NOs:431-682.

Features of the present disclosure include a method of modulatingsite-specific transcription within a target DNA, the method comprisingcontacting the target DNA with: (i) a DNA-targeting RNA, or a DNApolynucleotide encoding the same, wherein the DNA-targeting RNAcomprises: (a) a first segment comprising a nucleotide sequence that iscomplementary to a sequence in the target DNA; and (b) a second segmentthat interacts with a site-directed modifying polypeptide; and (ii) asite-directed modifying polypeptide, or a polynucleotide encoding thesame, wherein the site-directed modifying polypeptide comprises: (a) anRNA-binding portion that interacts with the DNA-targeting RNA; and (b)an activity portion that modulates transcription, wherein saidcontacting results in modulating transcription within the target DNA. Insome cases, transcription within the target DNA is increased. In somecases, transcription within the target DNA is decreased.

Features of the present disclosure include a method of site-specificmodification at target DNA, the method comprising: contacting the targetDNA with: (i) a DNA-targeting RNA, or a DNA polynucleotide encoding thesame, wherein the DNA-targeting RNA comprises: (a) a first segmentcomprising a nucleotide sequence that is complementary to a sequence inthe target DNA; and (b) a second segment that interacts with asite-directed modifying polypeptide; and (ii) a site-directed modifyingpolypeptide, or a polynucleotide encoding the same, wherein thesite-directed modifying polypeptide comprises: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that modulates transcription within the target DNA. In somecases, the site-directed modifying polypeptide increases transcriptionwithin the target DNA. In some cases, the site-directed modifyingpolypeptide decreases transcription within the target DNA.

Features of the present disclosure include a method of promotingsite-specific cleavage and modification of a target DNA in a cell, themethod comprising introducing into the cell: (i) a DNA-targeting RNA, ora DNA polynucleotide encoding the same, wherein the DNA-targeting RNAcomprises: (a) a first segment comprising a nucleotide sequence that iscomplementary to a sequence in the target DNA; and (b) a second segmentthat interacts with a site-directed modifying polypeptide; and (ii) asite-directed modifying polypeptide, or a polynucleotide encoding thesame, wherein the site-directed modifying polypeptide comprises: (a) anRNA-binding portion that interacts with the DNA-targeting RNA; and (b)an activity portion that exhibits nuclease activity that creates adouble strand break in the target DNA; wherein the site of the doublestrand break is determined by the DNA-targeting RNA, the contactingoccurs under conditions that are permissive for nonhomologous endjoining or homology-directed repair, and the target DNA is cleaved andrejoined to produce a modified DNA sequence. In some cases, the methodfurther comprises contacting the target DNA with a donor polynucleotide,wherein the donor polynucleotide, a portion of the donor polynucleotide,a copy of the donor polynucleotide, or a portion of a copy of the donorpolynucleotide integrates into the target DNA. In some cases, the methoddoes not comprise contacting the cell with a donor polynucleotide,wherein the target DNA is modified such that nucleotides within thetarget DNA are deleted. In some cases, the cell is selected from thegroup consisting of: an archaeal cell, a bacterial cell, a eukaryoticcell, a eukaryotic single-cell organism, a somatic cell, a germ cell, astem cell, a plant cell, an algal cell, an animal cell, in invertebratecell, a vertebrate cell, a fish cell, a frog cell, a bird cell, amammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, arodent cell, a rat cell, a mouse cell, a non-human primate cell, and ahuman cell. In some cases, the cell is in vitro. In some cases, the cellis in vivo.

Features of the present disclosure include a method of producing agenetically modified cell in a subject, the method comprising: (I)introducing into a cell: (i) a DNA-targeting RNA, or a DNApolynucleotide encoding the same, wherein the DNA-targeting RNAcomprises: (a) a first segment comprising a nucleotide sequence that iscomplementary to a sequence in the target DNA; and (b) a second segmentthat interacts with a site-directed modifying polypeptide; and (ii) asite-directed modifying polypeptide, or a polynucleotide encoding thesame, wherein the site-directed modifying polypeptide comprises: (a) anRNA-binding portion that interacts with the DNA-targeting RNA; and (b)an activity portion that exhibits nuclease activity that creates adouble strand break in the target DNA; wherein the site of the doublestrand break is determined by the DNA-targeting RNA, the contactingoccurs under conditions that are permissive for nonhomologous endjoining or homology-directed repair, and the target DNA is cleaved andrejoined to produce a modified DNA sequence; thereby producing thegenetically modified cell; and (II) transplanting the geneticallymodified cell into the subject. In some cases, the method furthercomprises contacting the cell with a donor polynucleotide, wherein thedonor polynucleotide, a portion of the donor polynucleotide, a copy ofthe donor polynucleotide, or a portion of a copy of the donorpolynucleotide integrates into the target DNA. In some cases, the methoddoes not comprise contacting the cell with a donor polynucleotide,wherein the target DNA is modified such that nucleotides within thetarget DNA are deleted. In some cases, the cell is selected from thegroup consisting of: an archaeal cell, a bacterial cell, a eukaryoticcell, a eukaryotic single-cell organism, a somatic cell, a germ cell, astem cell, a plant cell, an algal cell, an animal cell, in invertebratecell, a vertebrate cell, a fish cell, an amphibian cell, a bird cell, amammalian cell, an ungulate cell, a rodent cell, a non-human primatecell, and a human cell.

Features of the present disclosure include a method of modifying targetDNA in a genetically modified cell that comprises a nucleotide sequenceencoding an exogenous site-directed modifying polypeptide, the methodcomprising introducing into the genetically modified cell aDNA-targeting RNA, or a DNA polynucleotide encoding the same, wherein:(i) the DNA-targeting RNA comprises: (a) a first segment comprising anucleotide sequence that is complementary to a sequence in the targetDNA; and (b) a second segment that interacts with a site-directedmodifying polypeptide; and (ii) the site-directed modifying polypeptidecomprises: (a) an RNA-binding portion that interacts with theDNA-targeting RNA; and (b) an activity portion that exhibits nucleaseactivity. In some cases, the site-directed modifying polypeptidecomprises an amino acid sequence having at least about 75% amino acidsequence identity to amino acids 7-166 or 731-1003 of the Cas9/Csn1amino acid sequence depicted in FIG. 3A and FIG. 3B, or to thecorresponding portions in any of the amino acid sequences set forth asSEQ ID NOs:1-256 and 795-1346. In some cases, the cell is selected fromthe group consisting of: an archaeal cell, a bacterial cell, aeukaryotic cell, a eukaryotic single-cell organism, a somatic cell, agerm cell, a stem cell, a plant cell, an algal cell, an animal cell, ininvertebrate cell, a vertebrate cell, a fish cell, an amphibian cell, abird cell, a mammalian cell, an ungulate cell, a rodent cell, anon-human primate cell, and a human cell. In some cases, the cell is invivo. In some cases, the cell is in vitro. In some cases, the expressionof the site-directed modifying polypeptide is under the control of aninducible promoter. In some cases, the expression of the site-directedmodifying polypeptide is under the control of a cell type-specificpromoter.

Features of the present disclosure include a kit comprising: theDNA-targeting RNA, or a DNA polynucleotide encoding the same; and areagent for reconstitution and/or dilution. In some cases, the kitfurther comprises a reagent selected from the group consisting of: abuffer for introducing into cells the DNA-targeting RNA, a wash buffer,a control reagent, a control expression vector or RNA polynucleotide, areagent for transcribing the DNA-targeting RNA from DNA, andcombinations thereof.

Features of the present disclosure include a kit comprising: asite-directed modifying polypeptide of the present disclosure, or apolynucleotide encoding the same; and a reagent for reconstitutionand/or dilution. In some cases, the kit further comprises a reagentselected from the group consisting of: a buffer for introducing intocells the site-directed modifying polypeptide, a wash buffer, a controlreagent, a control expression vector or RNA polynucleotide, a reagentfor in vitro production of the site-directed modifying polypeptide fromDNA, and combinations thereof.

Features of the present disclosure include a kit comprising: asite-directed modifying polypeptide of the present disclosure, or apolynucleotide encoding the same; and a reagent for reconstitutionand/or dilution. Features of the present disclosure include a kitcomprising: a DNA-targeting RNA, or a DNA polynucleotide encoding thesame, the DNA-targeting RNA comprising: (a) a first segment comprising anucleotide sequence that is complementary to a sequence in a target DNA;and (b) a second segment that interacts with a site-directed modifyingpolypeptide; and (ii) the site-directed modifying polypeptide, or apolynucleotide encoding the same, the site-directed modifyingpolypeptide comprising: (a) an RNA-binding portion that interacts withthe DNA-targeting RNA; and (b) an activity portion that exhibitssite-directed enzymatic activity, wherein the site of enzymatic activityis determined by the DNA-targeting RNA.

Features of the present disclosure include a kit comprising: (i) aDNA-targeting RNA, or a DNA polynucleotide encoding the same,comprising: (a) a first segment comprising a nucleotide sequence that iscomplementary to a sequence in a target DNA; and (b) a second segmentthat interacts with a site-directed modifying polypeptide; and (ii) thesite-directed modifying polypeptide, or a polynucleotide encoding thesame, comprising: (a) an RNA-binding portion that interacts with theDNA-targeting RNA; and (b) an activity portion that that modulatestranscription within the target DNA, wherein the site of modulatedtranscription within the target DNA is determined by the DNA-targetingRNA.

Features of the present disclosure include a kit comprising: (i) any ofthe recombinant expression vectors above; and (ii) a reagent forreconstitution and/or dilution. Features of the present disclosureinclude a kit comprising: (i) any of the recombinant expression vectorsabove; and (ii) a recombinant expression vector comprising a nucleotidesequence that encodes a site-directed modifying polypeptide, wherein thesite-directed modifying polypeptide comprises: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that exhibits site-directed enzymatic activity, wherein the siteof enzymatic activity is determined by the DNA-targeting RNA. Featuresof the present disclosure include a kit comprising: (i) any of therecombinant expression vectors above; and (ii) a recombinant expressionvector comprising a nucleotide sequence that encodes a site-directedmodifying polypeptide, wherein the site-directed modifying polypeptidecomprises: (a) an RNA-binding portion that interacts with theDNA-targeting RNA; and (b) an activity portion that modulatestranscription within the target DNA, wherein the site of modulatedtranscription within the target DNA is determined by the DNA-targetingRNA.

Features of the present disclosure include a kit for targeting targetDNA comprising: two or more DNA-targeting RNAs, or DNA polynucleotidesencoding the same, wherein the first segment of at least one of the twoor more DNA-targeting RNAs differs by at least one nucleotide from thefirst segment of at least one other of the two or more DNA-targetingRNAs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B provide a schematic drawing of two exemplary subjectDNA-targeting RNAs, each associated with a site-directed modifyingpolypeptide and with a target DNA.

FIG. 2 depicts target DNA editing through double-stranded DNA breaksintroduced using a Cas9/Csn1 site-directed modifying polypeptide and aDNA-targeting RNA.

FIG. 3A-3B depict the amino acid sequence of a Cas9/Csn1 protein fromStreptococcus pyogenes (SEQ ID NO:8). Cas9 has domains homologous toboth HNH and RuvC endonucleases. (FIG. 3A) Motifs 1-4 are overlined(FIG. 3B) Domains 1 and 2 are overlined.

FIG. 4A-4B depict the percent identity between the Cas9/Csn1 proteinsfrom multiple species. (FIG. 4A) Sequence identity relative toStreptococcus pyogenes. For Example, Domain 1 is amino acids 7-166 andDomain 2 is amino acids 731-1003 of Cas9/Csn1 from Streptococcuspyogenes as depicted in FIG. 3B. (FIG. 4B) Sequence identity relative toNeisseria meningitidis. For example, Domain 1 is amino acids 13-139 andDomain 2 is amino acids 475-750 of Cas9/Csn1 from Neisseria meningitidis(SEQ ID NO:79).

FIG. 5 depicts a multiple sequence alignment of motifs 1-4 of Cas9/Csn1proteins from various diverse species selected from the phylogenetictable in FIG. 32 (see FIG. 32, FIG. 3A, and Table 1) (Streptococcuspyogenes (Motifs 1-4: SEQ ID NOs.: 1361-1364), Legionella pneumophila(Motifs 1-4: SEQ ID NOs.: 1365-1368), Gamma proteobacterium (Motifs 1-4:SEQ ID NOs.: 1369-1372), Listeria innocua (Motifs 1-4: SEQ ID NOs.:1373-1376), Lactobacillus gasseri (Motifs 1-4: SEQ ID NOs.: 1377-1380),Eubacterium rectale (Motifs 1-4: SEQ ID NOs.: 1381-1384), Staphylococcuslugdunensis (Motifs 1-4: SEQ ID NOs.: 1385-1388), Mycoplasma synoviae(Motifs 1-4: SEQ ID NOs.: 1389-1392), Mycoplasma mobile (Motifs 1-4: SEQID NOs.: 1393-1396), Wolinella succinogenes (Motifs 1-4: SEQ ID NOs.:1397-1400), Flavobacterium columnare (Motifs 1-4: SEQ ID NOs.:1401-1404), Fibrobacter succinogenes (Motifs 1-4: SEQ ID NOs.:1405-1408), Bacteroides fragilis (Motifs 1-4: SEQ ID NOs.: 1409-1412),Acidothermus cellulolyticus (Motifs 1-4: SEQ ID NOs.: 1413-1416), andBifidobacterium dentium (Motifs 1-4: SEQ ID NOs.: 1417-1420).

FIG. 6A-6B provide alignments of naturally occurring tracrRNA(“activator-RNA”) sequences from various species (L. innocua (SEQ ID NO:434); S. pyogenes (SEQ ID NO: 433); S. mutans (SEQ ID NO: 435); S.thermophilus1 (SEQ ID NO: 436); M. mobile (SEQ ID NO: 440); N.meningitides (SEQ ID NO: 438); P. multocida (SEQ ID NO: 439); S.thermophilus2 (SEQ ID NO: 437); and S. pyogenes (SEQ ID NO: 433). (FIG.6A) multiple sequence alignment of selected tracrRNA orthologues(AlignX, VectorNTI package, Invitrogen) associated with CRISPR/Cas lociof similar architecture and highly similar Cas9/Csn1 sequences. Blackboxes represent shared nucleotides (FIG. 6B) multiple sequence alignmentof selected tracrRNA orthologues (AlignX, VectorNTI package, Invitrogen)associated with CRISPR/Cas loci of different architecture andnon-closely related Cas9/Csn1 sequences. Note the sequence similarity ofN. meningitidis and P. multocida tracrRNA orthologues. Black boxesrepresent shared nucleotides. For more exemplary activator-RNAsequences, see SEQ ID NOs:431-562.

FIG. 7A-7B provide alignments of naturally occurring duplex-formingsegments of crRNA (“targeter-RNA”) sequences from various species (L.innocua (SEQ ID NO:577); S. pyogenes (SEQ ID NO:569); S. mutans (SEQ IDNO:574); S. thermophilus1 (SEQ ID NO:575); C. jejuni (SEQ ID NO:597); S.pyogenes (SEQ ID NO:569); F. novicida (SEQ ID NO:572); M. mobile (SEQ IDNO:571); N. meningitides (SEQ ID NO:579); P. multocida (SEQ ID NO:570);and S. thermophilus2 (SEQ ID NO:576). (FIG. 7A) multiple sequencealignments of exemplary duplex-forming segment of targeter-RNA sequences(AlignX, VectorNTI package, Invitrogen) associated with the loci ofsimilar architecture and highly similar Cas9/Csn1 sequences. (FIG. 7B)multiple sequence alignments of exemplary duplex-forming segment oftargeter-RNA sequences (AlignX, VectorNTI package, Invitrogen)associated with the loci of different architecture and diverse Cas9sequences. Black boxes represent shared nucleotides. For more exemplaryduplex-forming segments targeter-RNA sequences, see SEQ ID NOs:563-679.

FIG. 8 provides a schematic of hybridization for naturally occurringduplex-forming segments of the crRNA (“targeter-RNA”) with theduplex-forming segment of the corresponding tracrRNA orthologue(“activator-RNA”). Upper sequence, targeter-RNA; lower sequence,duplex-forming segment of the corresponding activator-RNA. The CRISPRloci belong to the Type II (Nmeni/CASS4) CRISPR/Cas system. Nomenclatureis according to the CRISPR database (CRISPR DB). SEQ ID numbers arelisted top to bottom: S. pyogenes (SEQ ID NOs:569 and 442); S. mutans(SEQ ID NOs:574 and 443); S. thermophilus1 (SEQ ID NOs:575 and 444); S.thermophilus2 (SEQ ID NOs:576 and 445); L. innocua (SEQ ID NOs:577 and446); T. denticola (SEQ ID NOs:578 and 448); N. meningitides (SEQ IDNOs:579 and 449); S. gordonii (SEQ ID NOs:580 and 451); B. bifidum (SEQID NOs:581 and 452); L. salivarius (SEQ ID NOs:582 and 453); F.tularensis (SEQ ID NOs:583, 454, 584, and 455); and L. pneumophila (SEQID NOs:585 and 456). Note that some species contain each two Type IICRISPR loci. For more exemplary activator-RNA sequences, see SEQ IDNOs:431-562. For more exemplary duplex-forming segments targeter-RNAsequences, see SEQ ID NOs:563-679.

FIG. 9 depicts example tracrRNA (activator-RNA) and crRNA (targeter-RNA)sequences from two species. A degree of interchangeability exists; forexample, the S. pyogenes Cas9/Csn1 protein is functional with tracrRNAand crRNA derived from L. innocua. (I) denotes a canonical Watson-Crickbase pair while (⋅) denotes a G-U wobble base pair. “Variable 20 nt” or“20 nt” represents the DNA-targeting segment that is complementary to atarget DNA (this region can be up to about 100 nt in length). Also shownis the design of single-molecule DNA-targeting RNA that incorporatesfeatures of the targeter-RNA and the activator-RNA. (Cas9/Csn1 proteinsequences from a wide variety of species are depicted in FIG. 3A andFIG. 3B and set forth as SEQ ID NOs:1-256 and 795-1346) Streptococcuspyogenes: top to bottom: (SEQ ID NOs: 1421, 478, 1423); Listeriainnocua: top to bottom: (SEQ ID NOs: 1422, 479, 1424). The sequencesprovided are non-limiting examples and are meant to illustrate howsingle-molecule DNA-targeting RNAs and two-molecule DNA-targeting RNAscan be designed based on naturally existing sequences from a widevariety of species. Various examples of suitable seuqences from a widevariety of species are set forth as follows (Cas9 protein: SEQ IDNOs:1-259; tracrRNAs: SEQ ID NOs:431-562, or the complements thereof;crRNAs: SEQ ID NOs:563-679, or the complements thereof; and examplesingle-molecule DNA-targeting RNAs: SEQ ID NOs:680-682).

FIG. 10A-10E show that Cas9 is a DNA endonuclease guided by two RNAmolecules. FIG. 10E (top to bottom, SEQ ID NOs: 278-280, and 431).

FIG. 11A-11B demonstrate that Cas9 uses two nuclease domains to cleavethe two strands in the target DNA.

FIG. 12A-12E illustrate that Cas9-catalyzed cleavage of target DNArequires an activating domain in tracrRNA and is governed by a seedsequence in the crRNA. FIG. 12C (top to bottom, SEQ ID NO:278-280, and431); FIG. 12D (top to bottom, SEQ ID NOs: 281-290); and FIG. 12E (topto bottom, SEQ ID NOs: 291-292, 283, 293-298).

FIG. 13A-13C show that a PAM is required to license target DNA cleavageby the Cas9-tracrRNA:crRNA complex.

FIG. 14A-14C illustrate that Cas9 can be programmed using a singleengineered RNA molecule combining tracrRNA and crRNA features. Chimera A(SEQ ID NO:299); Chimera B (SEQ ID NO:300).

FIG. 15 depicts the type II RNA-mediated CRISPR/Cas immune pathway.

FIG. 16A-16B depict purification of Cas9 nucleases.

FIG. 17A-17C show that Cas9 guided by dual-tracrRNA:crRNA cleavesprotospacer plasmid and oligonucleotide DNA. FIG. 17B (top to bottom,SEQ ID NOs: 301-303, and 487); and FIG. 17C (top to bottom, SEQ IDNO:304-306, and 431).

FIG. 18A-18B show that Cas9 is a Mg2+-dependent endonuclease with 3′-5′exonuclease activity.

FIG. 19A-19C illustrate that dual-tracrRNA:crRNA directed Cas9 cleavageof target DNA is site specific. FIG. 19A (top to bottom, SEQ IS NOs:1350 and 1351) FIG. 19C (top to bottom, SEQ ID NOs: 307-309, 487,337-339, and 431).

FIG. 20A-20B show that dual-tracrRNA:crRNA directed Cas9 cleavage oftarget DNA is fast and efficient.

FIG. 21A-21B show that the HNH and RuvC-like domains of Cas9 directcleavage of the complementary and noncomplementary DNA strand,respectively.

FIG. 22 demonstrates that tracrRNA is required for target DNArecognition.

FIG. 23A-23B show that a minimal region of tracrRNA is capable ofguiding dualtracrRNA:crRNA directed cleavage of target DNA.

FIG. 24A-24D demonstrate that dual-tracrRNA:crRNA guided target DNAcleavage by Cas9 can be species specific.

FIG. 25A-25C show that a seed sequence in the crRNA governs dualtracrRNA:crRNA directed cleavage of target DNA by Cas9. FIG. 25A: targetDNA probe 1 (SEQ ID NO:310); spacer 4 crRNA (1-42) (SEQ ID NO:311);tracrRNA (15-89) (SEQ ID NO: 1352). FIG. 25B left panel (SEQ ID NO:310).

FIG. 26A-26C demonstrate that the PAM sequence is essential forprotospacer plasmid DNA cleavage by Cas9-tracrRNA:crRNA and forCas9-mediated plasmid DNA interference in bacterial cells. FIG. 26B (topto bottom, SEQ ID NOs:312-314); and FIG. 26C (top to bottom, SEQ IDNO:315-320).

FIG. 27A-27C show that Cas9 guided by a single chimeric RNA mimickingdual tracrRNA:crRNA cleaves protospacer DNA. FIG. 27C (top to bottom,SEQ ID NO:321-324).

FIG. 28A-28D depict de novo design of chimeric RNAs targeting the GreenFluorescent Protein (GFP) gene sequence. FIG. 28B (top to bottom, SEQ IDNOs:325-326). FIG. 28C: GFP1 target sequence (SEQ ID NO:327); GFP2target sequence (SEQ ID NO:328); GFP3 target sequence (SEQ ID NO:329);GFP4 target sequence (SEQ ID NO:330); GFP5 target sequence (SEQ IDNO:331); GFP1 chimeric RNA (SEQ ID NO:332); GFP2 chimeric RNA (SEQ IDNO:333); GFP3 chimeric RNA (SEQ ID NO:334); GFP4 chimeric RNA (SEQ IDNO:335); GFP5 chimeric RNA (SEQ ID NO:336).

FIG. 29A-29E demonstrate that co-expression of Cas9 and guide RNA inhuman cells generates double-strand DNA breaks at the target locus. FIG.29C (top to bottom, SEQ ID NO:425-428).

FIG. 30A-30B demonstrate that cell lysates contain active Cas9:sgRNA andsupport site-specific DNA cleavage.

FIG. 31A-31B demonstrate that 3′ extension of sgRNA constructs enhancessite-specific NHEJ-mediated mutagenesis. FIG. 31A (top to bottom, SEQ IDNO:428-430).

FIG. 32A-32B depict a phylogenetic tree of representative Cas9 sequencesfrom various organisms (FIG. 32A) as well as Cas9 locus architecturesfor the main groups of the tree (FIG. 32B).

FIG. 33A-33E depict the architecture of type II CRISPR-Cas from selectedbacterial species.

FIG. 34A-34B depict tracrRNA and pre-crRNA co-processing in selectedtype II CRISPR Cas systems. FIG. 34A (top to bottom, SEQ ID NO: 618,442, 574, 443, 577, 447, 573, 481); FIG. 34B (top to bottom, SEQ ID NO:598, 470, 579, 450).

FIG. 35 depicts a sequence alignment of tracrRNA orthologuesdemonstrating the diversity of tracrRNA sequences.

FIG. 36A-36F depict the expression of bacterial tracrRNA orthologues andcrRNAs revealed by deep RNA sequencing.

FIG. 37A-37O list all tracrRNA orthologues and mature crRNAs retrievedby sequencing for the bacterial species studied, including coordinates(region of interest) and corresponding cDNA sequences (5′ to 3′).

FIG. 38A-38B present a table of bacterial species containing type IICRISPR-Cas loci characterized by the presence of the signature genecas9. These sequences were used for phylogenetic analyses.

FIG. 39A-39B depict the design of the CRISPR interference (CRISPRi)system.

FIG. 40A-40E demonstrate that CRISPRi effectively silences transcriptionelongation and initiation.

FIG. 41A-41B demonstrate that CRISPRi functions by blockingtranscription elongation.

FIG. 42A-42C demonstrate the targeting specificity of the CRISPRisystem.

FIG. 43A-43F depict the characterization of factors that affectsilencing efficiency.

FIG. 44A-44C depict functional profiling of a complex regulatory networkusing CRISPRi gene knockdown.

FIG. 45A-45B demonstrates gene silencing using CRISPRi in mammaliancells.

FIG. 46 depicts the mechanism of the type II CRISPR system from S.pyogenes.

FIG. 47A-47B depict the growth curves of E. coli cell culturesco-transformed with dCas9 and sgRNA.

FIG. 48 shows that CRISPRi could silence expression of a reporter geneon a multiple-copy plasmid.

FIG. 49A-49C depict the RNA-seq data of cells with sgRNAs that targetdifferent genes.

FIG. 50A-50E depict the silencing effects of sgRNAs with adjacent doublemismatches.

FIG. 51A-51C depict the combinatorial silencing effects of using twosgRNAs to regulate a single gene.

FIG. 52 shows that sgRNA repression is dependent on the target loci andrelatively distance from the transcription start.

FIG. 53A-53C depict experimental results demonstrating that a variantCas9 site-directed polypeptide (dCas9) is works for the subject methodswhen dCas9 has reduced activity in the RuvC1 domain only (e.g., D10A),the HNH domain only (e.g., H840A), or both domains (e.g, D10A andH840A).

FIG. 54A-54C list examples of suitable fusion partners (or fragmentsthereof) for a subject variant Cas9 site-directed polypeptide. Examplesinclude, but are not limited to those listed.

FIG. 55A-55D demonstrate that a chimeric site-directed polypeptide canbe used to activate (increase) transcription in human cells.

FIG. 56 demonstrates that a chimeric site-directed polypeptide can beused to repress (decrease) transcription in human cells.

FIG. 57A-57B demonstrate that artificial sequences that share roughly50% identity with naturally occurring a tracrRNAs and crRNAs canfunction with Cas9 to cleave target DNA as long as the structure of theprotein-binding domain of the DNA-targeting RNA is conserved.

DEFINITIONS—PART I

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases. “Oligonucleotide”generally refers to polynucleotides of between about 5 and about 100nucleotides of single- or double-stranded DNA. However, for the purposesof this disclosure, there is no upper limit to the length of anoligonucleotide. Oligonucleotides are also known as “oligomers” or“oligos” and may be isolated from genes, or chemically synthesized bymethods known in the art. The terms “polynucleotide” and “nucleic acid”should be understood to include, as applicable to the embodiments beingdescribed, single-stranded (such as sense or antisense) anddouble-stranded polynucleotides.

A “stem-loop structure” refers to a nucleic acid having a secondarystructure that includes a region of nucleotides which are known orpredicted to form a double strand (step portion) that is linked on oneside by a region of predominantly single-stranded nucleotides (loopportion). The terms “hairpin” and “fold-back” structures are also usedherein to refer to stem-loop structures. Such structures are well knownin the art and these terms are used consistently with their knownmeanings in the art. As is known in the art, a stem-loop structure doesnot require exact base-pairing. Thus, the stem may include one or morebase mismatches. Alternatively, the base-pairing may be exact, i.e. notinclude any mismatches.

By “hybridizable” or “complementary” or “substantially complementary” itis meant that a nucleic acid (e.g. RNA) comprises a sequence ofnucleotides that enables it to non-covalently bind, i.e. formWatson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,”to another nucleic acid in a sequence-specific, antiparallel, manner(i.e., a nucleic acid specifically binds to a complementary nucleicacid) under the appropriate in vitro and/or in vivo conditions oftemperature and solution ionic strength. As is known in the art,standard Watson-Crick base-pairing includes: adenine (A) pairing withthymidine (T), adenine (A) pairing with uracil (U), and guanine (G)pairing with cytosine (C) [DNA, RNA]. In addition, it is also known inthe art that for hybridization between two RNA molecules (e.g., dsRNA),guanine (G) base pairs with uracil (U). For example, G/U base-pairing ispartially responsible for the degeneracy (i.e., redundancy) of thegenetic code in the context of tRNA anti-codon base-pairing with codonsin mRNA. In the context of this disclosure, a guanine (G) of aprotein-binding segment (dsRNA duplex) of a subject DNA-targeting RNAmolecule is considered complementary to a uracil (U), and vice versa. Assuch, when a G/U base-pair can be made at a given nucleotide position aprotein-binding segment (dsRNA duplex) of a subject DNA-targeting RNAmolecule, the position is not considered to be non-complementary, but isinstead considered to be complementary.

Hybridization and washing conditions are well known and exemplified inSambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1therein; and Sambrook, J. and Russell, W., Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor (2001). The conditions of temperature and ionicstrength determine the “stringency” of the hybridization.

Hybridization requires that the two nucleic acids contain complementarysequences, although mismatches between bases are possible. Theconditions appropriate for hybridization between two nucleic acidsdepend on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof complementation between two nucleotide sequences, the greater thevalue of the melting temperature (Tm) for hybrids of nucleic acidshaving those sequences. For hybridizations between nucleic acids withshort stretches of complementarity (e.g. complementarity over 35 orless, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or lessnucleotides) the position of mismatches becomes important (see Sambrooket al., supra, 11.7-11.8). Typically, the length for a hybridizablenucleic acid is at least about 10 nucleotides. Illustrative minimumlengths for a hybridizable nucleic acid are: at least about 15nucleotides; at least about 20 nucleotides; at least about 22nucleotides; at least about 25 nucleotides; and at least about 30nucleotides). Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the region ofcomplementation and the degree of complementation.

It is understood in the art that the sequence of polynucleotide need notbe 100% complementary to that of its target nucleic acid to bespecifically hybridizable or hybridizable. Moreover, a polynucleotidemay hybridize over one or more segments such that intervening oradjacent segments are not involved in the hybridization event (e.g., aloop structure or hairpin structure). A polynucleotide can comprise atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100% sequence complementarity to a target region within the targetnucleic acid sequence to which they are targeted. For example, anantisense nucleic acid in which 18 of 20 nucleotides of the antisensecompound are complementary to a target region, and would thereforespecifically hybridize, would represent 90 percent complementarity. Inthis example, the remaining noncomplementary nucleotides may beclustered or interspersed with complementary nucleotides and need not becontiguous to each other or to complementary nucleotides. Percentcomplementarity between particular stretches of nucleic acid sequenceswithin nucleic acids can be determined routinely using BLAST programs(basic local alignment search tools) and PowerBLAST programs known inthe art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang andMadden, Genome Res., 1997, 7, 649-656) or by using the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482-489).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

“Binding” as used herein (e.g. with reference to an RNA-binding domainof a polypeptide) refers to a non-covalent interaction betweenmacromolecules (e.g., between a protein and a nucleic acid). While in astate of non-covalent interaction, the macromolecules are said to be“associated” or “interacting” or “binding” (e.g., when a molecule X issaid to interact with a molecule Y, it is meant the molecule X binds tomolecule Y in a non-covalent manner). Not all components of a bindinginteraction need be sequence-specific (e.g., contacts with phosphateresidues in a DNA backbone), but some portions of a binding interactionmay be sequence-specific. Binding interactions are generallycharacterized by a dissociation constant (Kd) of less than 10⁻⁶ M, lessthan 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻¹⁰ M, lessthan 10⁻¹¹ M, less than 10⁻¹² M, less than 10⁻¹³ M, less than 10⁻¹⁴ M,or less than 10⁻¹⁵ M. “Affinity” refers to the strength of binding,increased binding affinity being correlated with a lower Kd.

By “binding domain” it is meant a protein domain that is able to bindnon-covalently to another molecule. A binding domain can bind to, forexample, a DNA molecule (a DNA-binding protein), an RNA molecule (anRNA-binding protein) and/or a protein molecule (a protein-bindingprotein). In the case of a protein domain-binding protein, it can bindto itself (to form homodimers, homotrimers, etc.) and/or it can bind toone or more molecules of a different protein or proteins.

The term “conservative amino acid substitution” refers to theinterchangeability in proteins of amino acid residues having similarside chains. For example, a group of amino acids having aliphatic sidechains consists of glycine, alanine, valine, leucine, and isoleucine; agroup of amino acids having aliphatic-hydroxyl side chains consists ofserine and threonine; a group of amino acids having amide containingside chains consisting of asparagine and glutamine; a group of aminoacids having aromatic side chains consists of phenylalanine, tyrosine,and tryptophan; a group of amino acids having basic side chains consistsof lysine, arginine, and histidine; a group of amino acids having acidicside chains consists of glutamate and aspartate; and a group of aminoacids having sulfur containing side chains consists of cysteine andmethionine. Exemplary conservative amino acid substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same, and inthe same relative position, when comparing the two sequences. Sequenceidentity can be determined in a number of different manners. Todetermine sequence identity, sequences can be aligned using variousmethods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT,etc.), available over the world wide web at sites includingncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/,ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See,e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

A DNA sequence that “encodes” a particular RNA is a DNA nucleic acidsequence that is transcribed into RNA. A DNA polynucleotide may encodean RNA (mRNA) that is translated into protein, or a DNA polynucleotidemay encode an RNA that is not translated into protein (e.g. tRNA, rRNA,or a DNA-targeting RNA; also called “non-coding” RNA or “ncRNA”).

A “protein coding sequence” or a sequence that encodes a particularprotein or polypeptide, is a nucleic acid sequence that is transcribedinto mRNA (in the case of DNA) and is translated (in the case of mRNA)into a polypeptide in vitro or in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ terminus (N-terminus) and atranslation stop nonsense codon at the 3′ terminus (C-terminus). Acoding sequence can include, but is not limited to, cDNA fromprokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryoticor eukaryotic DNA, and synthetic nucleic acids. A transcriptiontermination sequence will usually be located 3′ to the coding sequence.

As used herein, a “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of a downstream(3′ direction) coding or non-coding sequence. For purposes of definingthe present invention, the promoter sequence is bounded at its 3′terminus by the transcription initiation site and extends upstream (5′direction) to include the minimum number of bases or elements necessaryto initiate transcription at levels detectable above background. Withinthe promoter sequence will be found a transcription initiation site, aswell as protein binding domains responsible for the binding of RNApolymerase. Eukaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes. Various promoters, including induciblepromoters, may be used to drive the various vectors of the presentinvention.

A promoter can be a constitutively active promoter (i.e., a promoterthat is constitutively in an active/“ON” state), it may be an induciblepromoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”,is controlled by an external stimulus, e.g., the presence of aparticular temperature, compound, or protein), it may be a spatiallyrestricted promoter (i.e., transcriptional control element, enhancer,etc.)(e.g., tissue specific promoter, cell type specific promoter,etc.), and it may be a temporally restricted promoter (i.e., thepromoter is in the “ON” state or “OFF” state during specific stages ofembryonic development or during specific stages of a biological process,e.g., hair follicle cycle in mice).

Suitable promoters can be derived from viruses and can therefore bereferred to as viral promoters, or they can be derived from anyorganism, including prokaryotic or eukaryotic organisms. Suitablepromoters can be used to drive expression by any RNA polymerase (e.g.,pol I, pol II, pol III). Exemplary promoters include, but are notlimited to the SV40 early promoter, mouse mammary tumor virus longterminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP);a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE), a rous sarcomavirus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishiet al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), ahuman H1 promoter (H1), and the like.

Examples of inducible promoters include, but are not limited toT7 RNApolymerase promoter, T3 RNA polymerase promoter,Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter,lactose induced promoter, heat shock promoter, Tetracycline-regulatedpromoter, Steroid-regulated promoter, Metal-regulated promoter, estrogenreceptor-regulated promoter, etc. Inducible promoters can therefore beregulated by molecules including, but not limited to, doxycycline; RNApolymerase, e.g., T7 RNA polymerase; an estrogen receptor; an estrogenreceptor fusion; etc.

In some embodiments, the promoter is a spatially restricted promoter(i.e., cell type specific promoter, tissue specific promoter, etc.) suchthat in a multi-cellular organism, the promoter is active (i.e., “ON”)in a subset of specific cells. Spatially restricted promoters may alsobe referred to as enhancers, transcriptional control elements, controlsequences, etc. Any convenient spatially restricted promoter may be usedand the choice of suitable promoter (e.g., a brain specific promoter, apromoter that drives expression in a subset of neurons, a promoter thatdrives expression in the germline, a promoter that drives expression inthe lungs, a promoter that drives expression in muscles, a promoter thatdrives expression in islet cells of the pancreas, etc.) will depend onthe organism. For example, various spatially restricted promoters areknown for plants, flies, worms, mammals, mice, etc. Thus, a spatiallyrestricted promoter can be used to regulate the expression of a nucleicacid encoding a subject site-directed modifying polypeptide in a widevariety of different tissues and cell types, depending on the organism.Some spatially restricted promoters are also temporally restricted suchthat the promoter is in the “ON” state or “OFF” state during specificstages of embryonic development or during specific stages of abiological process (e.g., hair follicle cycle in mice).

For illustration purposes, examples of spatially restricted promotersinclude, but are not limited to, neuron-specific promoters,adipocyte-specific promoters, cardiomyocyte-specific promoters, smoothmuscle-specific promoters, photoreceptor-specific promoters, etc.Neuron-specific spatially restricted promoters include, but are notlimited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBLHSENO2, X51956); an aromatic amino acid decarboxylase (AADC) promoter; aneurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsinpromoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see,e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat.Med. 16(10):1161-1166); a serotonin receptor promoter (see, e.g.,GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh etal. (2009) Gene Ther 16:437; Sasaoka et al. (1992) Mol. Brain Res.16:274; Boundy et al. (1998) J. Neurosci. 18:9989; and Kaneda et al.(1991) Neuron 6:583-594); a GnRH promoter (see, e.g., Radovick et al.(1991) Proc. Natl. Acad. Sci. USA 88:3402-3406); an L7 promoter (see,e.g., Oberdick et al. (1990) Science 248:223-226); a DNMT promoter (see,e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); anenkephalin promoter (see, e.g., Comb et al. (1988) EMBO J.17:3793-3805); a myelin basic protein (MBP) promoter; aCa2+-calmodulin-dependent protein kinase II-alpha (CamKIIα) promoter(see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250;and Casanova et al. (2001) Genesis 31:37); a CMVenhancer/platelet-derived growth factor-β promoter (see, e.g., Liu etal. (2004) Gene Therapy 11:52-60); and the like.

Adipocyte-specific spatially restricted promoters include, but are notlimited to aP2 gene promoter/enhancer, e.g., a region from −5.4 kb to+21 bp of a human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol.138:1604; Ross et al. (1990) Proc. Natl. Acad. Sci. USA 87:9590; andPavjani et al. (2005) Nat. Med. 11:797); a glucose transporter-4 (GLUT4)promoter (see, e.g., Knight et al. (2003) Proc. Natl. Acad. Sci. USA100:14725); a fatty acid translocase (FAT/CD36) promoter (see, e.g.,Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato et al. (2002)J. Biol. Chem. 277:15703); a stearoyl-CoA desaturase-1 (SCD1) promoter(Tabor et al. (1999) J. Biol. Chem. 274:20603); a leptin promoter (see,e.g., Mason et al. (1998) Endocrinol. 139:1013; and Chen et al. (1999)Biochem. Biophys. Res. Comm. 262:187); an adiponectin promoter (see,e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm. 331:484; andChakrabarti (2010) Endocrinol. 151:2408); an adipsin promoter (see,e.g., Platt et al. (1989) Proc. Natl. Acad. Sci. USA 86:7490); aresistin promoter (see, e.g., Seo et al. (2003) Molec. Endocrinol.17:1522); and the like.

Cardiomyocyte-specific spatially restricted promoters include, but arenot limited to control sequences derived from the following genes:myosin light chain-2, α-myosin heavy chain, AE3, cardiac troponin C,cardiac actin, and the like. Franz et al. (1997) Cardiovasc. Res.35:560-566; Robbins et al. (1995) Ann. N.Y. Acad. Sci. 752:492-505; Linnet al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Mol. Cell.Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; andSartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.

Smooth muscle-specific spatially restricted promoters include, but arenot limited to an SM22a promoter (see, e.g., Akyürek et al. (2000) Mol.Med. 6:983; and U.S. Pat. No. 7,169,874); a smoothelin promoter (see,e.g., WO 2001/018048); an α-smooth muscle actin promoter; and the like.For example, a 0.4 kb region of the SM22a promoter, within which lie twoCArG elements, has been shown to mediate vascular smooth musclecell-specific expression (see, e.g., Kim, et al. (1997) Mol. Cell. Biol.17, 2266-2278; Li, et al., (1996) J. Cell Biol. 132, 849-859; andMoessler, et al. (1996) Development 122, 2415-2425).

Photoreceptor-specific spatially restricted promoters include, but arenot limited to, a rhodopsin promoter; a rhodopsin kinase promoter (Younget al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterasegene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); a retinitispigmentosa gene promoter (Nicoud et al. (2007) supra); aninterphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoudet al. (2007) supra); an IRBP gene promoter (Yokoyama et al. (1992) ExpEye Res. 55:225); and the like.

The terms “DNA regulatory sequences,” “control elements,” and“regulatory elements,” used interchangeably herein, refer totranscriptional and translational control sequences, such as promoters,enhancers, polyadenylation signals, terminators, protein degradationsignals, and the like, that provide for and/or regulate transcription ofa non-coding sequence (e.g., DNA-targeting RNA) or a coding sequence(e.g., site-directed modifying polypeptide, or Cas9/Csn1 polypeptide)and/or regulate translation of an encoded polypeptide.

The term “naturally-occurring” or “unmodified” as used herein as appliedto a nucleic acid, a polypeptide, a cell, or an organism, refers to anucleic acid, polypeptide, cell, or organism that is found in nature.For example, a polypeptide or polynucleotide sequence that is present inan organism (including viruses) that can be isolated from a source innature and which has not been intentionally modified by a human in thelaboratory is naturally occurring.

The term “chimeric” as used herein as applied to a nucleic acid orpolypeptide refers to two components that are defined by structuresderived from different sources. For example, where “chimeric” is used inthe context of a chimeric polypeptide (e.g., a chimeric Cas9/Csn1protein), the chimeric polypeptide includes amino acid sequences thatare derived from different polypeptides. A chimeric polypeptide maycomprise either modified or naturally-occurring polypeptide sequences(e.g., a first amino acid sequence from a modified or unmodifiedCas9/Csn1 protein; and a second amino acid sequence other than theCas9/Csn1 protein). Similarly, “chimeric” in the context of apolynucleotide encoding a chimeric polypeptide includes nucleotidesequences derived from different coding regions (e.g., a firstnucleotide sequence encoding a modified or unmodified Cas9/Csn1 protein;and a second nucleotide sequence encoding a polypeptide other than aCas9/Csn1 protein).

The term “chimeric polypeptide” refers to a polypeptide which is made bythe combination (i.e., “fusion”) of two otherwise separated segments ofamino sequence, usually through human intervention. A polypeptide thatcomprises a chimeric amino acid sequence is a chimeric polypeptide. Somechimeric polypeptides can be referred to as “fusion variants.”

“Heterologous,” as used herein, means a nucleotide or polypeptidesequence that is not found in the native nucleic acid or protein,respectively. For example, in a chimeric Cas9/Csn1 protein, theRNA-binding domain of a naturally-occurring bacterial Cas9/Csn1polypeptide (or a variant thereof) may be fused to a heterologouspolypeptide sequence (i.e. a polypeptide sequence from a protein otherthan Cas9/Csn1 or a polypeptide sequence from another organism). Theheterologous polypeptide sequence may exhibit an activity (e.g.,enzymatic activity) that will also be exhibited by the chimericCas9/Csn1 protein (e.g., methyltransferase activity, acetyltransferaseactivity, kinase activity, ubiquitinating activity, etc.). Aheterologous nucleic acid sequence may be linked to anaturally-occurring nucleic acid sequence (or a variant thereof) (e.g.,by genetic engineering) to generate a chimeric nucleotide sequenceencoding a chimeric polypeptide. As another example, in a fusion variantCas9 site-directed polypeptide, a variant Cas9 site-directed polypeptidemay be fused to a heterologous polypeptide (i.e. a polypeptide otherthan Cas9), which exhibits an activity that will also be exhibited bythe fusion variant Cas9 site-directed polypeptide. A heterologousnucleic acid sequence may be linked to a variant Cas9 site-directedpolypeptide (e.g., by genetic engineering) to generate a nucleotidesequence encoding a fusion variant Cas9 site-directed polypeptide.

“Recombinant,” as used herein, means that a particular nucleic acid (DNAor RNA) is the product of various combinations of cloning, restriction,polymerase chain reaction (PCR) and/or ligation steps resulting in aconstruct having a structural coding or non-coding sequencedistinguishable from endogenous nucleic acids found in natural systems.DNA sequences encoding polypeptides can be assembled from cDNA fragmentsor from a series of synthetic oligonucleotides, to provide a syntheticnucleic acid which is capable of being expressed from a recombinanttranscriptional unit contained in a cell or in a cell-free transcriptionand translation system. Genomic DNA comprising the relevant sequencescan also be used in the formation of a recombinant gene ortranscriptional unit. Sequences of non-translated DNA may be present 5′or 3′ from the open reading frame, where such sequences do not interferewith manipulation or expression of the coding regions, and may indeedact to modulate production of a desired product by various mechanisms(see “DNA regulatory sequences”, below). Alternatively, DNA sequencesencoding RNA (e.g., DNA-targeting RNA) that is not translated may alsobe considered recombinant. Thus, e.g., the term “recombinant” nucleicacid refers to one which is not naturally occurring, e.g., is made bythe artificial combination of two otherwise separated segments ofsequence through human intervention. This artificial combination isoften accomplished by either chemical synthesis means, or by theartificial manipulation of isolated segments of nucleic acids, e.g., bygenetic engineering techniques. Such is usually done to replace a codonwith a codon encoding the same amino acid, a conservative amino acid, ora non-conservative amino acid. Alternatively, it is performed to jointogether nucleic acid segments of desired functions to generate adesired combination of functions. This artificial combination is oftenaccomplished by either chemical synthesis means, or by the artificialmanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques. When a recombinant polynucleotide encodes apolypeptide, the sequence of the encoded polypeptide can be naturallyoccurring (“wild type”) or can be a variant (e.g., a mutant) of thenaturally occurring sequence. Thus, the term “recombinant” polypeptidedoes not necessarily refer to a polypeptide whose sequence does notnaturally occur. Instead, a “recombinant” polypeptide is encoded by arecombinant DNA sequence, but the sequence of the polypeptide can benaturally occurring (“wild type”) or non-naturally occurring (e.g., avariant, a mutant, etc.). Thus, a “recombinant” polypeptide is theresult of human intervention, but may be a naturally occurring aminoacid sequence.

A “vector” or “expression vector” is a replicon, such as plasmid, phage,virus, or cosmid, to which another DNA segment, i.e. an “insert”, may beattached so as to bring about the replication of the attached segment ina cell.

An “expression cassette” comprises a DNA coding sequence operably linkedto a promoter. “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. For instance, a promoter is operablylinked to a coding sequence if the promoter affects its transcription orexpression.

The terms “recombinant expression vector,” or “DNA construct” are usedinterchangeably herein to refer to a DNA molecule comprising a vectorand at least one insert. Recombinant expression vectors are usuallygenerated for the purpose of expressing and/or propagating theinsert(s), or for the construction of other recombinant nucleotidesequences. The insert(s) may or may not be operably linked to a promotersequence and may or may not be operably linked to DNA regulatorysequences.

A cell has been “genetically modified” or “transformed” or “transfected”by exogenous DNA, e.g. a recombinant expression vector, when such DNAhas been introduced inside the cell. The presence of the exogenous DNAresults in permanent or transient genetic change. The transforming DNAmay or may not be integrated (covalently linked) into the genome of thecell. In prokaryotes, yeast, and mammalian cells for example, thetransforming DNA may be maintained on an episomal element such as aplasmid. With respect to eukaryotic cells, a stably transformed cell isone in which the transforming DNA has become integrated into achromosome so that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones that comprise apopulation of daughter cells containing the transforming DNA. A “clone”is a population of cells derived from a single cell or common ancestorby mitosis. A “cell line” is a clone of a primary cell that is capableof stable growth in vitro for many generations.

Suitable methods of genetic modification (also referred to as“transformation”) include e.g., viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, calcium phosphate precipitation, polyethyleneimine(PEI)-mediated transfection, DEAE-dextran mediated transfection,liposome-mediated transfection, particle gun technology, calciumphosphate precipitation, direct micro injection, nanoparticle-mediatednucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev.2012 Sep. 13. pii: S0169-409X(12)00283-9. doi:10.1016/j.addr.2012.09.023), and the like.

The choice of method of genetic modification is generally dependent onthe type of cell being transformed and the circumstances under which thetransformation is taking place (e.g., in vitro, ex vivo, or in vivo). Ageneral discussion of these methods can be found in Ausubel, et al.,Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

A “target DNA” as used herein is a DNA polynucleotide that comprises a“target site” or “target sequence.” The terms “target site” or “targetsequence” or “target protospacer DNA” are used interchangeably herein torefer to a nucleic acid sequence present in a target DNA to which aDNA-targeting segment of a subject DNA-targeting RNA will bind (see FIG.1A-1B and FIG. 39A-39B), provided sufficient conditions for bindingexist. For example, the target site (or target sequence) 5′-GAGCATATC-3′within a target DNA is targeted by (or is bound by, or hybridizes with,or is complementary to) the RNA sequence 5′-GAUAUGCUC-3′. SuitableDNA/RNA binding conditions include physiological conditions normallypresent in a cell. Other suitable DNA/RNA binding conditions (e.g.,conditions in a cell-free system) are known in the art; see, e.g.,Sambrook, supra. The strand of the target DNA that is complementary toand hybridizes with the DNA-targeting RNA is referred to as the“complementary strand” and the strand of the target DNA that iscomplementary to the “complementary strand” (and is therefore notcomplementary to the DNA-targeting RNA) is referred to as the“noncomplementary strand” or “non-complementary strand” (see FIG.12A-12E).

By “site-directed modifying polypeptide” or “RNA-binding site-directedpolypeptide” or “RNA-binding site-directed modifying polypeptide” or“site-directed polypeptide” it is meant a polypeptide that binds RNA andis targeted to a specific DNA sequence. A site-directed modifyingpolypeptide as described herein is targeted to a specific DNA sequenceby the RNA molecule to which it is bound. The RNA molecule comprises asequence that is complementary to a target sequence within the targetDNA, thus targeting the bound polypeptide to a specific location withinthe target DNA (the target sequence).

By “cleavage” it is meant the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, a complex comprising a DNA-targeting RNA and asite-directed modifying polypeptide is used for targeted double-strandedDNA cleavage.

“Nuclease” and “endonuclease” are used interchangeably herein to mean anenzyme which possesses catalytic activity for DNA cleavage.

By “cleavage domain” or “active domain” or “nuclease domain” of anuclease it is meant the polypeptide sequence or domain within thenuclease which possesses the catalytic activity for DNA cleavage. Acleavage domain can be contained in a single polypeptide chain orcleavage activity can result from the association of two (or more)polypeptides. A single nuclease domain may consist of more than oneisolated stretch of amino acids within a given polypeptide.

The RNA molecule that binds to the site-directed modifying polypeptideand targets the polypeptide to a specific location within the target DNAis referred to herein as the “DNA-targeting RNA” or “DNA-targeting RNApolynucleotide” (also referred to herein as a “guide RNA” or “gRNA”). Asubject DNA-targeting RNA comprises two segments, a “DNA-targetingsegment” and a “protein-binding segment.” By “segment” it is meant asegment/section/region of a molecule, e.g., a contiguous stretch ofnucleotides in an RNA. A segment can also mean a region/section of acomplex such that a segment may comprise regions of more than onemolecule. For example, in some cases the protein-binding segment(described below) of a DNA-targeting RNA is one RNA molecule and theprotein-binding segment therefore comprises a region of that RNAmolecule. In other cases, the protein-binding segment (described below)of a DNA-targeting RNA comprises two separate molecules that arehybridized along a region of complementarity. As an illustrative,non-limiting example, a protein-binding segment of a DNA-targeting RNAthat comprises two separate molecules can comprise (i) base pairs 40-75of a first RNA molecule that is 100 base pairs in length; and (ii) basepairs 10-25 of a second RNA molecule that is 50 base pairs in length.The definition of “segment,” unless otherwise specifically defined in aparticular context, is not limited to a specific number of total basepairs, is not limited to any particular number of base pairs from agiven RNA molecule, is not limited to a particular number of separatemolecules within a complex, and may include regions of RNA moleculesthat are of any total length and may or may not include regions withcomplementarity to other molecules.

The DNA-targeting segment (or “DNA-targeting sequence”) comprises anucleotide sequence that is complementary to a specific sequence withina target DNA (the complementary strand of the target DNA). Theprotein-binding segment (or “protein-binding sequence”) interacts with asite-directed modifying polypeptide. When the site-directed modifyingpolypeptide is a Cas9 or Cas9 related polypeptide (described in moredetail below), site-specific cleavage of the target DNA occurs atlocations determined by both (i) base-pairing complementarity betweenthe DNA-targeting RNA and the target DNA; and (ii) a short motif(referred to as the protospacer adjacent motif (PAM)) in the target DNA.

The protein-binding segment of a subject DNA-targeting RNA comprises twocomplementary stretches of nucleotides that hybridize to one another toform a double stranded RNA duplex (dsRNA duplex).

In some embodiments, a subject nucleic acid (e.g., a DNA-targeting RNA,a nucleic acid comprising a nucleotide sequence encoding a DNA-targetingRNA; a nucleic acid encoding a site-directed polypeptide; etc.)comprises a modification or sequence that provides for an additionaldesirable feature (e.g., modified or regulated stability; subcellulartargeting; tracking, e.g., a fluorescent label; a binding site for aprotein or protein complex; etc.). Non-limiting examples include: a 5′cap (e.g., a 7-methylguanylate cap (m7G)); a 3′ polyadenylated tail(i.e., a 3′ poly(A) tail); a riboswitch sequence (e.g., to allow forregulated stability and/or regulated accessibility by proteins and/orprotein complexes); a stability control sequence; a sequence that formsa dsRNA duplex (i.e., a hairpin)); a modification or sequence thattargets the RNA to a subcellular location (e.g., nucleus, mitochondria,chloroplasts, and the like); a modification or sequence that providesfor tracking (e.g., direct conjugation to a fluorescent molecule,conjugation to a moiety that facilitates fluorescent detection, asequence that allows for fluorescent detection, etc.); a modification orsequence that provides a binding site for proteins (e.g., proteins thatact on DNA, including transcriptional activators, transcriptionalrepressors, DNA methyltransferases, DNA demethylases, histoneacetyltransferases, histone deacetylases, and the like); andcombinations thereof.

In some embodiments, a DNA-targeting RNA comprises an additional segmentat either the 5′ or 3′ end that provides for any of the featuresdescribed above. For example, a suitable third segment can comprise a 5′cap (e.g., a 7-methylguanylate cap (m7G)); a 3′ polyadenylated tail(i.e., a 3′ poly(A) tail); a riboswitch sequence (e.g., to allow forregulated stability and/or regulated accessibility by proteins andprotein complexes); a stability control sequence; a sequence that formsa dsRNA duplex (i.e., a hairpin)); a sequence that targets the RNA to asubcellular location (e.g., nucleus, mitochondria, chloroplasts, and thelike); a modification or sequence that provides for tracking (e.g.,direct conjugation to a fluorescent molecule, conjugation to a moietythat facilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.); a modification or sequence that provides abinding site for proteins (e.g., proteins that act on DNA, includingtranscriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like); and combinations thereof.

A subject DNA-targeting RNA and a subject site-directed modifyingpolypeptide (i.e., site-directed polypeptide) form a complex (i.e., bindvia non-covalent interactions). The DNA-targeting RNA provides targetspecificity to the complex by comprising a nucleotide sequence that iscomplementary to a sequence of a target DNA. The site-directed modifyingpolypeptide of the complex provides the site-specific activity. In otherwords, the site-directed modifying polypeptide is guided to a target DNAsequence (e.g. a target sequence in a chromosomal nucleic acid; a targetsequence in an extrachromosomal nucleic acid, e.g. an episomal nucleicacid, a minicircle, etc.; a target sequence in a mitochondrial nucleicacid; a target sequence in a chloroplast nucleic acid; a target sequencein a plasmid; etc.) by virtue of its association with theprotein-binding segment of the DNA-targeting RNA.

In some embodiments, a subject DNA-targeting RNA comprises two separateRNA molecules (RNA polynucleotides: an “activator-RNA” and a“targeter-RNA”, see below) and is referred to herein as a“double-molecule DNA-targeting RNA” or a “two-molecule DNA-targetingRNA.” In other embodiments, the subject DNA-targeting RNA is a singleRNA molecule (single RNA polynucleotide) and is referred to herein as a“single-molecule DNA-targeting RNA,” a “single-guide RNA,” or an“sgRNA.” The term “DNA-targeting RNA” or “gRNA” is inclusive, referringboth to double-molecule DNA-targeting RNAs and to single-moleculeDNA-targeting RNAs (i.e., sgRNAs).

An exemplary two-molecule DNA-targeting RNA comprises a crRNA-like(“CRISPR RNA” or “targeter-RNA” or “crRNA” or “crRNA repeat”) moleculeand a corresponding tracrRNA-like (“trans-acting CRISPR RNA” or“activator-RNA” or “tracrRNA”) molecule. A crRNA-like molecule(targeter-RNA) comprises both the DNA-targeting segment (singlestranded) of the DNA-targeting RNA and a stretch (“duplex-formingsegment”) of nucleotides that forms one half of the dsRNA duplex of theprotein-binding segment of the DNA-targeting RNA. A correspondingtracrRNA-like molecule (activator-RNA) comprises a stretch ofnucleotides (duplex-forming segment) that forms the other half of thedsRNA duplex of the protein-binding segment of the DNA-targeting RNA. Inother words, a stretch of nucleotides of a crRNA-like molecule arecomplementary to and hybridize with a stretch of nucleotides of atracrRNA-like molecule to form the dsRNA duplex of the protein-bindingdomain of the DNA-targeting RNA. As such, each crRNA-like molecule canbe said to have a corresponding tracrRNA-like molecule. The crRNA-likemolecule additionally provides the single stranded DNA-targetingsegment. Thus, a crRNA-like and a tracrRNA-like molecule (as acorresponding pair) hybridize to form a DNA-targeting RNA. The exactsequence of a given crRNA or tracrRNA molecule is characteristic of thespecies in which the RNA molecules are found. Various crRNAs andtracrRNAs are depicted in corresponding complementary pairs in FIG. 8. Asubject double-molecule DNA-targeting RNA can comprise any correspondingcrRNA and tracrRNA pair. A subject double-molecule DNA-targeting RNA cancomprise any corresponding crRNA and tracrRNA pair.

The term “activator-RNA” is used herein to mean a tracrRNA-like moleculeof a double-molecule DNA-targeting RNA. The term “targeter-RNA” is usedherein to mean a crRNA-like molecule of a double-molecule DNA-targetingRNA. The term “duplex-forming segment” is used herein to mean thestretch of nucleotides of an activator-RNA or a targeter-RNA thatcontributes to the formation of the dsRNA duplex by hybridizing to astretch of nucleotides of a corresponding activator-RNA or targeter-RNAmolecule. In other words, an activator-RNA comprises a duplex-formingsegment that is complementary to the duplex-forming segment of thecorresponding targeter-RNA. As such, an activator-RNA comprises aduplex-forming segment while a targeter-RNA comprises both aduplex-forming segment and the DNA-targeting segment of theDNA-targeting RNA. Therefore, a subject double-molecule DNA-targetingRNA can be comprised of any corresponding activator-RNA and targeter-RNApair.

A “host cell,” as used herein, denotes an in vivo or in vitro eukaryoticcell, a prokaryotic cell (e.g., bacterial or archaeal cell), or a cellfrom a multicellular organism (e.g., a cell line) cultured as aunicellular entity, which eukaryotic or prokaryotic cells can be, orhave been, used as recipients for a nucleic acid, and include theprogeny of the original cell which has been transformed by the nucleicacid. It is understood that the progeny of a single cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement as the original parent, due to natural, accidental, ordeliberate mutation. A “recombinant host cell” (also referred to as a“genetically modified host cell”) is a host cell into which has beenintroduced a heterologous nucleic acid, e.g., an expression vector. Forexample, a subject bacterial host cell is a genetically modifiedbacterial host cell by virtue of introduction into a suitable bacterialhost cell of an exogenous nucleic acid (e.g., a plasmid or recombinantexpression vector) and a subject eukaryotic host cell is a geneticallymodified eukaryotic host cell (e.g., a mammalian germ cell), by virtueof introduction into a suitable eukaryotic host cell of an exogenousnucleic acid.

The term “stem cell” is used herein to refer to a cell (e.g., plant stemcell, vertebrate stem cell) that has the ability both to self-renew andto generate a differentiated cell type (see Morrison et al. (1997) Cell88:287-298). In the context of cell ontogeny, the adjective“differentiated”, or “differentiating” is a relative term. A“differentiated cell” is a cell that has progressed further down thedevelopmental pathway than the cell it is being compared with. Thus,pluripotent stem cells (described below) can differentiate intolineage-restricted progenitor cells (e.g., mesodermal stem cells), whichin turn can differentiate into cells that are further restricted (e.g.,neuron progenitors), which can differentiate into end-stage cells (i.e.,terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.),which play a characteristic role in a certain tissue type, and may ormay not retain the capacity to proliferate further. Stem cells may becharacterized by both the presence of specific markers (e.g., proteins,RNAs, etc.) and the absence of specific markers. Stem cells may also beidentified by functional assays both in vitro and in vivo, particularlyassays relating to the ability of stem cells to give rise to multipledifferentiated progeny.

Stem cells of interest include pluripotent stem cells (PSCs). The term“pluripotent stem cell” or “PSC” is used herein to mean a stem cellcapable of producing all cell types of the organism. Therefore, a PSCcan give rise to cells of all germ layers of the organism (e.g., theendoderm, mesoderm, and ectoderm of a vertebrate). Pluripotent cells arecapable of forming teratomas and of contributing to ectoderm, mesoderm,or endoderm tissues in a living organism. Pluripotent stem cells ofplants are capable of giving rise to all cell types of the plant (e.g.,cells of the root, stem, leaves, etc.).

PSCs of animals can be derived in a number of different ways. Forexample, embryonic stem cells (ESCs) are derived from the inner cellmass of an embryo (Thomson et. al, Science. 1998 Nov. 6;282(5391):1145-7) whereas induced pluripotent stem cells (iPSCs) arederived from somatic cells (Takahashi et. al, Cell. 2007 Nov. 30;131(5):861-72; Takahashi et. al, Nat Protoc. 2007; 2(12):3081-9; Yu et.al, Science. 2007 Dec. 21; 318(5858):1917-20. Epub 2007 Nov. 20).Because the term PSC refers to pluripotent stem cells regardless oftheir derivation, the term PSC encompasses the terms ESC and iPSC, aswell as the term embryonic germ stem cells (EGSC), which are anotherexample of a PSC. PSCs may be in the form of an established cell line,they may be obtained directly from primary embryonic tissue, or they maybe derived from a somatic cell. PSCs can be target cells of the methodsdescribed herein.

By “embryonic stem cell” (ESC) is meant a PSC that was isolated from anembryo, typically from the inner cell mass of the blastocyst. ESC linesare listed in the NIH Human Embryonic Stem Cell Registry, e.g.hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1,HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1(MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (Universityof California at San Francisco); and H1, H7, H9, H13, H14 (WisconsinAlumni Research Foundation (WiCell Research Institute)). Stem cells ofinterest also include embryonic stem cells from other primates, such asRhesus stem cells and marmoset stem cells. The stem cells may beobtained from any mammalian species, e.g. human, equine, bovine,porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc.(Thomson et al. (1998) Science 282:1145; Thomson et al. (1995) Proc.Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254;Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Inculture, ESCs typically grow as flat colonies with largenucleo-cytoplasmic ratios, defined borders and prominent nucleoli. Inaddition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and AlkalinePhosphatase, but not SSEA-1. Examples of methods of generating andcharacterizing ESCs may be found in, for example, U.S. Pat. Nos.7,029,913, 5,843,780, and 6,200,806, the disclosures of which areincorporated herein by reference. Methods for proliferating hESCs in theundifferentiated form are described in WO 99/20741, WO 01/51616, and WO03/020920.

By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EGcell” is meant a PSC that is derived from germ cells and/or germ cellprogenitors, e.g. primordial germ cells, i.e. those that would becomesperm and eggs. Embryonic germ cells (EG cells) are thought to haveproperties similar to embryonic stem cells as described above. Examplesof methods of generating and characterizing EG cells may be found in,for example, U.S. Pat. No. 7,153,684; Matsui, Y., et al., (1992) Cell70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113;Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; andKoshimizu, U., et al. (1996) Development, 122:1235, the disclosures ofwhich are incorporated herein by reference.

By “induced pluripotent stem cell” or “iPSC” it is meant a PSC that isderived from a cell that is not a PSC (i.e., from a cell this isdifferentiated relative to a PSC). iPSCs can be derived from multipledifferent cell types, including terminally differentiated cells. iPSCshave an ES cell-like morphology, growing as flat colonies with largenucleo-cytoplasmic ratios, defined borders and prominent nuclei. Inaddition, iPSCs express one or more key pluripotency markers known byone of ordinary skill in the art, including but not limited to AlkalinePhosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1,Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods ofgenerating and characterizing iPSCs may be found in, for example, U.S.Patent Publication Nos. US20090047263, US20090068742, US20090191159,US20090227032, US20090246875, and US20090304646, the disclosures ofwhich are incorporated herein by reference. Generally, to generateiPSCs, somatic cells are provided with reprogramming factors (e.g. Oct4,SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram thesomatic cells to become pluripotent stem cells.

By “somatic cell” it is meant any cell in an organism that, in theabsence of experimental manipulation, does not ordinarily give rise toall types of cells in an organism. In other words, somatic cells arecells that have differentiated sufficiently that they will not naturallygenerate cells of all three germ layers of the body, i.e. ectoderm,mesoderm and endoderm. For example, somatic cells would include bothneurons and neural progenitors, the latter of which may be able tonaturally give rise to all or some cell types of the central nervoussystem but cannot give rise to cells of the mesoderm or endodermlineages.

By “mitotic cell” it is meant a cell undergoing mitosis. Mitosis is theprocess by which a eukaryotic cell separates the chromosomes in itsnucleus into two identical sets in two separate nuclei. It is generallyfollowed immediately by cytokinesis, which divides the nuclei,cytoplasm, organelles and cell membrane into two cells containingroughly equal shares of these cellular components.

By “post-mitotic cell” it is meant a cell that has exited from mitosis,i.e., it is “quiescent”, i.e. it is no longer undergoing divisions. Thisquiescent state may be temporary, i.e. reversible, or it may bepermanent.

By “meiotic cell” it is meant a cell that is undergoing meiosis. Meiosisis the process by which a cell divides its nuclear material for thepurpose of producing gametes or spores. Unlike mitosis, in meiosis, thechromosomes undergo a recombination step which shuffles genetic materialbetween chromosomes. Additionally, the outcome of meiosis is four(genetically unique) haploid cells, as compared with the two(genetically identical) diploid cells produced from mitosis.

By “recombination” it is meant a process of exchange of geneticinformation between two polynucleotides. As used herein,“homology-directed repair (HDR)” refers to the specialized form DNArepair that takes place, for example, during repair of double-strandbreaks in cells. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and leads to thetransfer of genetic information from the donor to the target.Homology-directed repair may result in an alteration of the sequence ofthe target molecule (e.g., insertion, deletion, mutation), if the donorpolynucleotide differs from the target molecule and part or all of thesequence of the donor polynucleotide is incorporated into the targetDNA. In some embodiments, the donor polynucleotide, a portion of thedonor polynucleotide, a copy of the donor polynucleotide, or a portionof a copy of the donor polynucleotide integrates into the target DNA.

By “non-homologous end joining (NHEJ)” it is meant the repair ofdouble-strand breaks in DNA by direct ligation of the break ends to oneanother without the need for a homologous template (in contrast tohomology-directed repair, which requires a homologous sequence to guiderepair). NHEJ often results in the loss (deletion) of nucleotidesequence near the site of the double-strand break.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease or symptom in a mammal, and includes:(a) preventing the disease or symptom from occurring in a subject whichmay be predisposed to acquiring the disease or symptom but has not yetbeen diagnosed as having it; (b) inhibiting the disease or symptom,i.e., arresting its development; or (c) relieving the disease, i.e.,causing regression of the disease. The therapeutic agent may beadministered before, during or after the onset of disease or injury. Thetreatment of ongoing disease, where the treatment stabilizes or reducesthe undesirable clinical symptoms of the patient, is of particularinterest. Such treatment is desirably performed prior to complete lossof function in the affected tissues. The subject therapy will desirablybe administered during the symptomatic stage of the disease, and in somecases after the symptomatic stage of the disease.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that as used herein and in the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “apolynucleotide” includes a plurality of such polynucleotides andreference to “the polypeptide” includes reference to one or morepolypeptides and equivalents thereof known to those skilled in the art,and so forth. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION—PART I

The present disclosure provides a DNA-targeting RNA that comprises atargeting sequence and, together with a modifying polypeptide, providesfor site-specific modification of a target DNA and/or a polypeptideassociated with the target DNA. The present disclosure further providessite-specific modifying polypeptides. The present disclosure furtherprovides methods of site-specific modification of a target DNA and/or apolypeptide associated with the target DNA The present disclosureprovides methods of modulating transcription of a target nucleic acid ina target cell, generally involving contacting the target nucleic acidwith an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA.Kits and compositions for carrying out the methods are also provided.The present disclosure provides genetically modified cells that produceCas9; and Cas9 transgenic non-human multicellular organisms.

Nucleic Acids

DNA-Targeting RNA

The present disclosure provides a DNA-targeting RNA that directs theactivities of an associated polypeptide (e.g., a site-directed modifyingpolypeptide) to a specific target sequence within a target DNA. Asubject DNA-targeting RNA comprises: a first segment (also referred toherein as a “DNA-targeting segment” or a “DNA-targeting sequence”) and asecond segment (also referred to herein as a “protein-binding segment”or a “protein-binding sequence”).

DNA-Targeting Segment of a DNA-Targeting RNA

The DNA-targeting segment of a subject DNA-targeting RNA comprises anucleotide sequence that is complementary to a sequence in a target DNA.In other words, the DNA-targeting segment of a subject DNA-targeting RNAinteracts with a target DNA in a sequence-specific manner viahybridization (i.e., base pairing). As such, the nucleotide sequence ofthe DNA-targeting segment may vary and determines the location withinthe target DNA that the DNA-targeting RNA and the target DNA willinteract. The DNA-targeting segment of a subject DNA-targeting RNA canbe modified (e.g., by genetic engineering) to hybridize to any desiredsequence within a target DNA.

The DNA-targeting segment can have a length of from about 12 nucleotidesto about 100 nucleotides. For example, the DNA-targeting segment canhave a length of from about 12 nucleotides (nt) to about 80 nt, fromabout 12 nt to about 50 nt, from about 12 nt to about 40 nt, from about12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 ntto about 20 nt, or from about 12 nt to about 19 nt. For example, theDNA-targeting segment can have a length of from about 19 nt to about 20nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt,from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, fromabout 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about19 nt to about 60 nt, from about 19 nt to about 70 nt, from about 19 ntto about 80 nt, from about 19 nt to about 90 nt, from about 19 nt toabout 100 nt, from about 20 nt to about 25 nt, from about 20 nt to about30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt,from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, fromabout 20 nt to about 60 nt, from about 20 nt to about 70 nt, from about20 nt to about 80 nt, from about 20 nt to about 90 nt, or from about 20nt to about 100 nt. The nucleotide sequence (the DNA-targeting sequence)of the DNA-targeting segment that is complementary to a nucleotidesequence (target sequence) of the target DNA can have a length at leastabout 12 nt. For example, the DNA-targeting sequence of theDNA-targeting segment that is complementary to a target sequence of thetarget DNA can have a length at least about 12 nt, at least about 15 nt,at least about 18 nt, at least about 19 nt, at least about 20 nt, atleast about 25 nt, at least about 30 nt, at least about 35 nt or atleast about 40 nt. For example, the DNA-targeting sequence of theDNA-targeting segment that is complementary to a target sequence of thetarget DNA can have a length of from about 12 nucleotides (nt) to about80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt,from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, fromabout 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 ntto about 20 nt, from about 19 nt to about 25 nt, from about 19 nt toabout 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt,from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, fromabout 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 ntto about 50 nt, or from about 20 nt to about 60 nt. The nucleotidesequence (the DNA-targeting sequence) of the DNA-targeting segment thatis complementary to a nucleotide sequence (target sequence) of thetarget DNA can have a length at least about 12 nt.

In some cases, the DNA-targeting sequence of the DNA-targeting segmentthat is complementary to a target sequence of the target DNA is 20nucleotides in length. In some cases, the DNA-targeting sequence of theDNA-targeting segment that is complementary to a target sequence of thetarget DNA is 19 nucleotides in length.

The percent complementarity between the DNA-targeting sequence of theDNA-targeting segment and the target sequence of the target DNA can beat least 60% (e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, at least 99%, or 100%). In some cases, the percent complementaritybetween the DNA-targeting sequence of the DNA-targeting segment and thetarget sequence of the target DNA is 100% over the seven contiguous5′-most nucleotides of the target sequence of the complementary strandof the target DNA. In some cases, the percent complementarity betweenthe DNA-targeting sequence of the DNA-targeting segment and the targetsequence of the target DNA is at least 60% over about 20 contiguousnucleotides. In some cases, the percent complementarity between theDNA-targeting sequence of the DNA-targeting segment and the targetsequence of the target DNA is 100% over the fourteen contiguous 5′-mostnucleotides of the target sequence of the complementary strand of thetarget DNA and as low as 0% over the remainder. In such a case, theDNA-targeting sequence can be considered to be 14 nucleotides in length(see FIG. 12D-12E). In some cases, the percent complementarity betweenthe DNA-targeting sequence of the DNA-targeting segment and the targetsequence of the target DNA is 100% over the seven contiguous 5′-mostnucleotides of the target sequence of the complementary strand of thetarget DNA and as low as 0% over the remainder. In such a case, theDNA-targeting sequence can be considered to be 7 nucleotides in length.

Protein-Binding Segment of a DNA-Targeting RNA

The protein-binding segment of a subject DNA-targeting RNA interactswith a site-directed modifying polypeptide. The subject DNA-targetingRNA guides the bound polypeptide to a specific nucleotide sequencewithin target DNA via the above mentioned DNA-targeting segment. Theprotein-binding segment of a subject DNA-targeting RNA comprises twostretches of nucleotides that are complementary to one another. Thecomplementary nucleotides of the protein-binding segment hybridize toform a double stranded RNA duplex (dsRNA) (see FIGS. 1A and 1B).

A subject double-molecule DNA-targeting RNA comprises two separate RNAmolecules. Each of the two RNA molecules of a subject double-moleculeDNA-targeting RNA comprises a stretch of nucleotides that arecomplementary to one another such that the complementary nucleotides ofthe two RNA molecules hybridize to form the double stranded RNA duplexof the protein-binding segment (FIG. 1A).

In some embodiments, the duplex-forming segment of the activator-RNA isat least about 60% identical to one of the activator-RNA (tracrRNA)molecules set forth in SEQ ID NOs:431-562, or a complement thereof, overa stretch of at least 8 contiguous nucleotides. For example, theduplex-forming segment of the activator-RNA (or the DNA encoding theduplex-forming segment of the activator-RNA) is at least about 60%identical, at least about 65% identical, at least about 70% identical,at least about 75% identical, at least about 80% identical, at leastabout 85% identical, at least about 90% identical, at least about 95%identical, at least about 98% identical, at least about 99% identical,or 100% identical, to one of the tracrRNA sequences set forth in SEQ IDNOs:431-562, or a complement thereof, over a stretch of at least 8contiguous nucleotides.

In some embodiments, the duplex-forming segment of the targeter-RNA isat least about 60% identical to one of the targeter-RNA (crRNA)sequences set forth in SEQ ID NOs:563-679, or a complement thereof, overa stretch of at least 8 contiguous nucleotides. For example, theduplex-forming segment of the targeter-RNA (or the DNA encoding theduplex-forming segment of the targeter-RNA) is at least about 65%identical, at least about 70% identical, at least about 75% identical,at least about 80% identical, at least about 85% identical, at leastabout 90% identical, at least about 95% identical, at least about 98%identical, at least about 99% identical or 100% identical to one of thecrRNA sequences set forth in SEQ ID NOs:563-679, or a complementthereof, over a stretch of at least 8 contiguous nucleotides.

A two-molecule DNA-targeting RNA can be designed to allow for controlled(i.e., conditional) binding of a targeter-RNA with an activator-RNA.Because a two-molecule DNA-targeting RNA is not functional unless boththe activator-RNA and the targeter-RNA are bound in a functional complexwith dCas9, a two-molecule DNA-targeting RNA can be inducible (e.g.,drug inducible) by rendering the binding between the activator-RNA andthe targeter-RNA to be inducible. As one non-limiting example, RNAaptamers can be used to regulate (i.e., control) the binding of theactivator-RNA with the targeter-RNA. Accordingly, the activator-RNAand/or the targeter-RNA can comprise an RNA aptamer sequence.

RNA aptamers are known in the art and are generally a synthetic versionof a riboswitch. The terms “RNA aptamer” and “riboswitch” are usedinterchangeably herein to encompass both synthetic and natural nucleicacid sequences that provide for inducible regulation of the structure(and therefore the availability of specific sequences) of the RNAmolecule of which they are part. RNA aptamers usually comprise asequence that folds into a particular structure (e.g., a hairpin), whichspecifically binds a particular drug (e.g., a small molecule). Bindingof the drug causes a structural change in the folding of the RNA, whichchanges a feature of the nucleic acid of which the aptamer is a part. Asnon-limiting examples: (i) an activator-RNA with an aptamer may not beable to bind to the cognate targeter-RNA unless the aptamer is bound bythe appropriate drug; (ii) a targeter-RNA with an aptamer may not beable to bind to the cognate activator-RNA unless the aptamer is bound bythe appropriate drug; and (iii) a targeter-RNA and an activator-RNA,each comprising a different aptamer that binds a different drug, may notbe able to bind to each other unless both drugs are present. Asillustrated by these examples, a two-molecule DNA-targeting RNA can bedesigned to be inducible.

Examples of aptamers and riboswitches can be found, for example, in:Nakamura et al., Genes Cells. 2012 May; 17(5):344-64; Vavalle et al.,Future Cardiol. 2012 May; 8(3):371-82; Citartan et al., BiosensBioelectron. 2012 Apr. 15; 34(1):1-11; and Liberman et al., WileyInterdiscip Rev RNA. 2012 May-June; 3(3):369-84; all of which are hereinincorporated by reference in their entirety.

Non-limiting examples of nucleotide sequences that can be included in atwo-molecule DNA-targeting RNA include either of the sequences set forthin SEQ ID NOs:431-562, or complements thereof pairing with any sequencesset forth in SEQ ID NOs:563-679, or complements thereof that canhybridize to form a protein binding segment.

A subject single-molecule DNA-targeting RNA comprises two stretches ofnucleotides (a targeter-RNA and an activator-RNA) that are complementaryto one another, are covalently linked by intervening nucleotides(“linkers” or “linker nucleotides”), and hybridize to form the doublestranded RNA duplex (dsRNA duplex) of the protein-binding segment, thusresulting in a stem-loop structure (FIG. 1B). The targeter-RNA and theactivator-RNA can be covalently linked via the 3′ end of thetargeter-RNA and the 5′ end of the activator-RNA. Alternatively,targeter-RNA and the activator-RNA can be covalently linked via the 5′end of the targeter-RNA and the 3′ end of the activator-RNA.

The linker of a single-molecule DNA-targeting RNA can have a length offrom about 3 nucleotides to about 100 nucleotides. For example, thelinker can have a length of from about 3 nucleotides (nt) to about 90nt, from about 3 nucleotides (nt) to about 80 nt, from about 3nucleotides (nt) to about 70 nt, from about 3 nucleotides (nt) to about60 nt, from about 3 nucleotides (nt) to about 50 nt, from about 3nucleotides (nt) to about 40 nt, from about 3 nucleotides (nt) to about30 nt, from about 3 nucleotides (nt) to about 20 nt or from about 3nucleotides (nt) to about 10 nt. For example, the linker can have alength of from about 3 nt to about 5 nt, from about 5 nt to about 10 nt,from about 10 nt to about 15 nt, from about 15 nt to about 20 nt, fromabout 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about30 nt to about 35 nt, from about 35 nt to about 40 nt, from about 40 ntto about 50 nt, from about 50 nt to about 60 nt, from about 60 nt toabout 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about90 nt, or from about 90 nt to about 100 nt. In some embodiments, thelinker of a single-molecule DNA-targeting RNA is 4 nt.

An exemplary single-molecule DNA-targeting RNA comprises twocomplementary stretches of nucleotides that hybridize to form a dsRNAduplex. In some embodiments, one of the two complementary stretches ofnucleotides of the single-molecule DNA-targeting RNA (or the DNAencoding the stretch) is at least about 60% identical to one of theactivator-RNA (tracrRNA) molecules set forth in SEQ ID NOs:431-562, or acomplement thereof, over a stretch of at least 8 contiguous nucleotides.For example, one of the two complementary stretches of nucleotides ofthe single-molecule DNA-targeting RNA (or the DNA encoding the stretch)is at least about 65% identical, at least about 70% identical, at leastabout 75% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical, at least about 95% identical,at least about 98% identical, at least about 99% identical or 100%identical to one of the tracrRNA sequences set forth in SEQ IDNOs:431-562, or a complement thereof, over a stretch of at least 8contiguous nucleotides.

In some embodiments, one of the two complementary stretches ofnucleotides of the single-molecule DNA-targeting RNA (or the DNAencoding the stretch) is at least about 60% identical to one of thetargeter-RNA (crRNA) sequences set forth in SEQ ID NOs:563-679, or acomplement thereof, over a stretch of at least 8 contiguous nucleotides.For example, one of the two complementary stretches of nucleotides ofthe single-molecule DNA-targeting RNA (or the DNA encoding the stretch)is at least about 65% identical, at least about 70% identical, at leastabout 75% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical, at least about 95% identical,at least about 98% identical, at least about 99% identical or 100%identical to one of the crRNA sequences set forth in SEQ ID NOs:563-679,or a complement thereof, over a stretch of at least 8 contiguousnucleotides.

Appropriate naturally occurring cognate pairs of crRNAs and tracrRNAscan be routinely determined for SEQ ID NOs:431-679 by taking intoaccount the speices name and base-pairing (for the dsRNA duplex of theprotein-binding domain) when determining appropriate cognate pairs (seeFIG. 8 as a non-limiting example).

With regard to both a subject single-molecule DNA-targeting RNA and to asubject double-molecule DNA-targeting RNA, FIG. 57A-57B demonstratesthat artificial sequences that share very little (roughly 50% identity)with naturally occurring a tracrRNAs and crRNAs can function with Cas9to cleave target DNA as long as the structure of the protein-bindingdomain of the DNA-targeting RNA is conserved. Thus, RNA foldingstructure of a naturally ocuring protein-binding domain of aDNA-trageting RNA can be taken into account in order to designartificial protein-binding domains (either two-molecule orsingle-molecule versions). As a non-limiting example, the functionalartificial DNA-trageting RNA of FIG. 57A-57B was designed based on thestructure of the protein-binding segment of the naturally occurringDNA-targeting (e.g., including the same number of base pairs along theRNA duplex and including the same “buldge” region as present in thenaturally occurring RNA). As structures can readily be produced by oneof ordinary skill in the art for any naturally occurring crRNA:tracrRNApair from any speices (see SEQ ID NOs:431-679 for crRNA and tracrRNAsequences from a wide variety of species), an artificialDNA-targeting-RNA can be designed to mimic the natural structure for agiven species when using the Cas9 (or a related Cas9, see FIG. 32A) fromthat species. (see FIG. 24D and related details in Example 1). Thus, asuitable DNA-targeting RNA can be an artificially designed RNA(non-naturally occurring) comprising a protein-binding domain that wasdesigned to mimic the structure of a protein-binding domain of anaturally occurring DNA-targeting RNA. (see SEQ ID NOs:431-679, takinginto account the species name when determining appropriate cognatepairs).

The protein-binding segment can have a length of from about 10nucleotides to about 100 nucleotides. For example, the protein-bindingsegment can have a length of from about 15 nucleotides (nt) to about 80nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt.

Also with regard to both a subject single-molecule DNA-targeting RNA andto a subject double-molecule DNA-targeting RNA, the dsRNA duplex of theprotein-binding segment can have a length from about 6 base pairs (bp)to about 50 bp. For example, the dsRNA duplex of the protein-bindingsegment can have a length from about 6 bp to about 40 bp, from about 6bp to about 30 bp, from about 6 bp to about 25 bp, from about 6 bp toabout 20 bp, from about 6 bp to about 15 bp, from about 8 bp to about 40bp, from about 8 bp to about 30 bp, from about 8 bp to about 25 bp, fromabout 8 bp to about 20 bp or from about 8 bp to about 15 bp. Forexample, the dsRNA duplex of the protein-binding segment can have alength from about from about 8 bp to about 10 bp, from about 10 bp toabout 15 bp, from about 15 bp to about 18 bp, from about 18 bp to about20 bp, from about 20 bp to about 25 bp, from about 25 bp to about 30 bp,from about 30 bp to about 35 bp, from about 35 bp to about 40 bp, orfrom about 40 bp to about 50 bp. In some embodiments, the dsRNA duplexof the protein-binding segment has a length of 36 base pairs. Thepercent complementarity between the nucleotide sequences that hybridizeto form the dsRNA duplex of the protein-binding segment can be at leastabout 60%. For example, the percent complementarity between thenucleotide sequences that hybridize to form the dsRNA duplex of theprotein-binding segment can be at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or at least about99%. In some cases, the percent complementarity between the nucleotidesequences that hybridize to form the dsRNA duplex of the protein-bindingsegment is 100%.

Site-Directed Modifying Polypeptide

A subject DNA-targeting RNA and a subject site-directed modifyingpolypeptide form a complex. The DNA-targeting RNA provides targetspecificity to the complex by comprising a nucleotide sequence that iscomplementary to a sequence of a target DNA (as noted above). Thesite-directed modifying polypeptide of the complex provides thesite-specific activity. In other words, the site-directed modifyingpolypeptide is guided to a DNA sequence (e.g. a chromosomal sequence oran extrachromosomal sequence, e.g. an episomal sequence, a minicirclesequence, a mitochondrial sequence, a chloroplast sequence, etc.) byvirtue of its association with at least the protein-binding segment ofthe DNA-targeting RNA (described above).

A subject site-directed modifying polypeptide modifies target DNA (e.g.,cleavage or methylation of target DNA) and/or a polypeptide associatedwith target DNA (e.g., methylation or acetylation of a histone tail). Asite-directed modifying polypeptide is also referred to herein as a“site-directed polypeptide” or an “RNA binding site-directed modifyingpolypeptide.”

In some cases, the site-directed modifying polypeptide is anaturally-occurring modifying polypeptide. In other cases, thesite-directed modifying polypeptide is not a naturally-occurringpolypeptide (e.g., a chimeric polypeptide as discussed below or anaturally-occurring polypeptide that is modified, e.g., mutation,deletion, insertion).

Exemplary naturally-occurring site-directed modifying polypeptides areset forth in SEQ ID NOs:1-255 as a non-limiting and non-exhaustive listof naturally occurring Cas9/Csn1 endonucleases. These naturallyoccurring polypeptides, as disclosed herein, bind a DNA-targeting RNA,are thereby directed to a specific sequence within a target DNA, andcleave the target DNA to generate a double strand break. A subjectsite-directed modifying polypeptide comprises two portions, anRNA-binding portion and an activity portion. In some embodiments, asubject site-directed modifying polypeptide comprises: (i) anRNA-binding portion that interacts with a DNA-targeting RNA, wherein theDNA-targeting RNA comprises a nucleotide sequence that is complementaryto a sequence in a target DNA; and (ii) an activity portion thatexhibits site-directed enzymatic activity (e.g., activity for DNAmethylation, activity for DNA cleavage, activity for histoneacetylation, activity for histone methylation, etc.), wherein the siteof enzymatic activity is determined by the DNA-targeting RNA.

In other embodiments, a subject site-directed modifying polypeptidecomprises: (i) an RNA-binding portion that interacts with aDNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotidesequence that is complementary to a sequence in a target DNA; and (ii)an activity portion that modulates transcription within the target DNA(e.g., to increase or decrease transcription), wherein the site ofmodulated transcription within the target DNA is determined by theDNA-targeting RNA.

In some cases, a subject site-directed modifying polypeptide hasenzymatic activity that modifies target DNA (e.g., nuclease activity,methyltransferase activity, demethylase activity, DNA repair activity,DNA damage activity, deamination activity, dismutase activity,alkylation activity, depurination activity, oxidation activity,pyrimidine dimer forming activity, integrase activity, transposaseactivity, recombinase activity, polymerase activity, ligase activity,helicase activity, photolyase activity or glycosylase activity).

In other cases, a subject site-directed modifying polypeptide hasenzymatic activity that modifies a polypeptide (e.g., a histone)associated with target DNA (e.g., methyltransferase activity,demethylase activity, acetyltransferase activity, deacetylase activity,kinase activity, phosphatase activity, ubiquitin ligase activity,deubiquitinating activity, adenylation activity, deadenylation activity,SUMOylating activity, deSUMOylating activity, ribosylation activity,deribosylation activity, myristoylation activity or demyristoylationactivity).

Exemplary Site-Directed Modifying Polypeptides

In some cases, the site-directed modifying polypeptide comprises anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, or 100%, amino acid sequence identity to amino acids 7-166 or731-1003 of the Cas9/Csn1 amino acid sequence depicted in FIG. 3A andFIG. 3B, or to the corresponding portions in any of the amino acidsequences set forth as SEQ ID NOs:1-256 and 795-1346.

Nucleic Acid Modifications

In some embodiments, a subject nucleic acid (e.g., a DNA-targeting RNA)comprises one or more modifications, e.g., a base modification, abackbone modification, etc, to provide the nucleic acid with a new orenhanced feature (e.g., improved stability). As is known in the art, anucleoside is a base-sugar combination. The base portion of thenucleoside is normally a heterocyclic base. The two most common classesof such heterocyclic bases are the purines and the pyrimidines.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, the 3′, or the 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turn,the respective ends of this linear polymeric compound can be furtherjoined to form a circular compound, however, linear compounds aregenerally suitable. In addition, linear compounds may have internalnucleotide base complementarity and may therefore fold in a manner as toproduce a fully or partially double-stranded compound. Withinoligonucleotides, the phosphate groups are commonly referred to asforming the internucleoside backbone of the oligonucleotide. The normallinkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Modified Backbones and Modified Internucleoside Linkages

Examples of suitable nucleic acids containing modifications includenucleic acids containing modified backbones or non-naturalinternucleoside linkages. Nucleic acids (having modified backbonesinclude those that retain a phosphorus atom in the backbone and thosethat do not have a phosphorus atom in the backbone.

Suitable modified oligonucleotide backbones containing a phosphorus atomtherein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Suitable oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be a basic (the nucleobase is missing or has ahydroxyl group in place thereof). Various salts (such as, for example,potassium or sodium), mixed salts and free acid forms are also included.

In some embodiments, a subject nucleic acid comprises one or morephosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂-(known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). MMI type internucleoside linkages are disclosed inthe above referenced U.S. Pat. No. 5,489,677. Suitable amideinternucleoside linkages are disclosed in t U.S. Pat. No. 5,602,240.

Also suitable are nucleic acids having morpholino backbone structures asdescribed in, e.g., U.S. Pat. No. 5,034,506. For example, in someembodiments, a subject nucleic acid comprises a 6-membered morpholinoring in place of a ribose ring. In some of these embodiments, aphosphorodiamidate or other non-phosphodiester internucleoside linkagereplaces a phosphodiester linkage.

Suitable modified polynucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Mimetics

A subject nucleic acid can be a nucleic acid mimetic. The term “mimetic”as it is applied to polynucleotides is intended to includepolynucleotides wherein only the furanose ring or both the furanose ringand the internucleotide linkage are replaced with non-furanose groups,replacement of only the furanose ring is also referred to in the art asbeing a sugar surrogate. The heterocyclic base moiety or a modifiedheterocyclic base moiety is maintained for hybridization with anappropriate target nucleic acid. One such nucleic acid, a polynucleotidemimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA, thesugar-backbone of a polynucleotide is replaced with an amide containingbackbone, in particular an aminoethylglycine backbone. The nucleotidesare retained and are bound directly or indirectly to aza nitrogen atomsof the amide portion of the backbone.

One polynucleotide mimetic that has been reported to have excellenthybridization properties is a peptide nucleic acid (PNA). The backbonein PNA compounds is two or more linked aminoethylglycine units whichgives PNA an amide containing backbone. The heterocyclic base moietiesare bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative U.S. patents that describe thepreparation of PNA compounds include, but are not limited to: U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262.

Another class of polynucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. One class of linking groups has been selected to give anon-ionic oligomeric compound. The non-ionic morpholino-based oligomericcompounds are less likely to have undesired interactions with cellularproteins. Morpholino-based polynucleotides are non-ionic mimics ofoligonucleotides which are less likely to form undesired interactionswith cellular proteins (Dwaine A. Braasch and David R. Corey,Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotidesare disclosed in U.S. Pat. No. 5,034,506. A variety of compounds withinthe morpholino class of polynucleotides have been prepared, having avariety of different linking groups joining the monomeric subunits.

A further class of polynucleotide mimetic is referred to as cyclohexenylnucleic acids (CeNA). The furanose ring normally present in a DNA/RNAmolecule is replaced with a cyclohexenyl ring. CeNA DMT protectedphosphoramidite monomers have been prepared and used for oligomericcompound synthesis following classical phosphoramidite chemistry. Fullymodified CeNA oligomeric compounds and oligonucleotides having specificpositions modified with CeNA have been prepared and studied (see Wang etal., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general theincorporation of CeNA monomers into a DNA chain increases its stabilityof a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA andDNA complements with similar stability to the native complexes. Thestudy of incorporating CeNA structures into natural nucleic acidstructures was shown by NMR and circular dichroism to proceed with easyconformational adaptation.

A further modification includes Locked Nucleic Acids (LNAs) in which the2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ringthereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming abicyclic sugar moiety. The linkage can be a methylene (—CH₂—), groupbridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2(Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNA analogsdisplay very high duplex thermal stabilities with complementary DNA andRNA (Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradationand good solubility properties. Potent and nontoxic antisenseoligonucleotides containing LNAs have been described (Wahlestedt et al.,Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methylcytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

Modified Sugar Moieties

A subject nucleic acid can also include one or more substituted sugarmoieties. Suitable polynucleotides comprise a sugar substituent groupselected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C.sub.1 to C₁₀ alkyl or C₂ to C₁₀alkenyl and alkynyl. Particularly suitable are O((CH₂)_(n)O)_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from 1 to about 10. Othersuitable polynucleotides comprise a sugar substituent group selectedfrom: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Asuitable modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further suitablemodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other suitable sugar substituent groups include methoxy (—O—CH₃),aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups may be in thearabino (up) position or ribo (down) position. A suitable 2′-arabinomodification is 2′-F. Similar modifications may also be made at otherpositions on the oligomeric compound, particularly the 3′ position ofthe sugar on the 3′ terminal nucleoside or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligomeric compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar.

Base Modifications and Substitutions

A subject nucleic acid may also include nucleobase (often referred to inthe art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C═C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are useful for increasing the binding affinity of anoligomeric compound. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi et al., eds., AntisenseResearch and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) andare suitable base substitutions, e.g., when combined with2′-O-methoxyethyl sugar modifications.

Conjugates

Another possible modification of a subject nucleic acid involveschemically linking to the polynucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups include, but are notlimited to, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Suitable conjugate groupsinclude, but are not limited to, cholesterols, lipids, phospholipids,biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties include groups that improve uptake, enhanceresistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that enhance thepharmacokinetic properties include groups that improve uptake,distribution, metabolism or excretion of a subject nucleic acid.

Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

A conjugate may include a “Protein Transduction Domain” or PTD (alsoknown as a CPP—cell penetrating peptide), which may refer to apolypeptide, polynucleotide, carbohydrate, or organic or inorganiccompound that facilitates traversing a lipid bilayer, micelle, cellmembrane, organelle membrane, or vesicle membrane. A PTD attached toanother molecule, which can range from a small polar molecule to a largemacromolecule and/or a nanoparticle, facilitates the molecule traversinga membrane, for example going from extracellular space to intracellularspace, or cytosol to within an organelle. In some embodiments, a PTD iscovalently linked to the amino terminus of an exogenous polypeptide(e.g., a site-directed modifying polypeptide). In some embodiments, aPTD is covalently linked to the carboxyl terminus of an exogenouspolypeptide (e.g., a site-directed modifying polypeptide). In someembodiments, a PTD is covalently linked to a nucleic acid (e.g., aDNA-targeting RNA, a polynucleotide encoding a DNA-targeting RNA, apolynucleotide encoding a site-directed modifying polypeptide, etc.).Exemplary PTDs include but are not limited to a minimal undecapeptideprotein transduction domain (corresponding to residues 47-57 of HIV-1TAT comprising YGRKKRRQRRR; SEQ ID NO:264); a polyarginine sequencecomprising a number of arginines sufficient to direct entry into a cell(e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain(Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an DrosophilaAntennapedia protein transduction domain (Noguchi et al. (2003) Diabetes52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al.(2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000)Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ IDNO:265); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:266);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:267); and RQIKIWFQNRRMKWKK(SEQ ID NO:268). Exemplary PTDs include but are not limited to,YGRKKRRQRRR (SEQ ID NO:264), RKKRRQRRR (SEQ ID NO:269); an argininehomopolymer of from 3 arginine residues to 50 arginine residues;Exemplary PTD domain amino acid sequences include, but are not limitedto, any of the following: YGRKKRRQRRR (SEQ ID NO:264); RKKRRQRR (SEQ IDNO:270); YARAAARQARA (SEQ ID NO:271); THRLPRRRRRR (SEQ ID NO:272); andGGRRARRRRRR (SEQ ID NO:273). In some embodiments, the PTD is anactivatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June;1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”)connected via a cleavable linker to a matching polyanion (e.g., Glu9 or“E9”), which reduces the net charge to nearly zero and thereby inhibitsadhesion and uptake into cells. Upon cleavage of the linker, thepolyanion is released, locally unmasking the polyarginine and itsinherent adhesiveness, thus “activating” the ACPP to traverse themembrane.

Exemplary DNA-Targeting RNAs

In some embodiments, a suitable DNA-targeting RNA comprises two separateRNA polynucleotide molecules. The first of the two separate RNApolynucleotide molecules (the activator-RNA) comprises a nucleotidesequence having at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100% nucleotide sequence identity over a stretch of at least 8contiguous nucleotides to any one of the nucleotide sequences set forthin SEQ ID NOs:431-562, or complements thereof. The second of the twoseparate RNA polynucleotide molecules (the targeter-RNA) comprises anucleotide sequence having at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100% nucleotide sequence identity over a stretch ofat least 8 contiguous nucleotides to any one of the nucleotide sequencesset forth in SEQ ID NOs:563-679, or complements thereof.

In some embodiments, a suitable DNA-targeting RNA is a single RNApolynucleotide and comprises a first nucleotide sequence having at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100% nucleotidesequence identity over a stretch of at least 8 contiguous nucleotides toany one of the nucleotide sequences set forth in SEQ ID NOs:431-562 anda second nucleotide sequence having at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100% nucleotide sequence identity over astretch of at least 8 contiguous nucleotides to any one of thenucleotide sequences set forth in SEQ ID NOs: 463-679.

In some embodiments, the DNA-targeting RNA is a double-moleculeDNA-targeting RNA and the targeter-RNA comprises the sequence5′GUUUUAGAGCUA-3′ (SEQ ID NO:679) linked at its 5′ end to a stretch ofnucleotides that are complementary to a target DNA. In some embodiments,the DNA-targeting RNA is a double-molecule DNA-targeting RNA and theactivator-RNA comprises the sequence 5′ UAGCAAGUUAAAAUAAGGCUAGUCCG-3′(SEQ ID NO: 397).

In some embodiments, the DNA-targeting RNA is a single-moleculeDNA-targeting RNA and comprises the sequence5′-GUUUUAGAGCUA-linker-UAGCAAGUUAAAAUAAGGCUAGUCCG-3′ linked at its 5′end to a stretch of nucleotides that are complementary to a target DNA(where “linker” denotes any a linker nucleotide sequence that cancomprise any nucleotide sequence) (SEQ ID NO: 680). Other exemplarysingle-molecule DNA-targeting RNAs include those set forth in SEQ IDNOs: 680-682.

Nucleic Acids Encoding a Subject DNA-Targeting RNA and/or a SubjectSite-Directed Modifying Polypeptide

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a subject DNA-targeting RNA and/or a subjectsite-directed modifying polypeptide. In some embodiments, a subjectDNA-targeting RNA-encoding nucleic acid is an expression vector, e.g., arecombinant expression vector.

In some embodiments, a subject method involves contacting a target DNAor introducing into a cell (or a population of cells) one or morenucleic acids comprising nucleotide sequences encoding a DNA-targetingRNA and/or a site-directed modifying polypeptide. In some embodiments acell comprising a target DNA is in vitro. In some embodiments a cellcomprising a target DNA is in vivo. Suitable nucleic acids comprisingnucleotide sequences encoding a DNA-targeting RNA and/or a site-directedmodifying polypeptide include expression vectors, where an expressionvector comprising a nucleotide sequence encoding a DNA-targeting RNAand/or a site-directed modifying polypeptide is a “recombinantexpression vector.”

In some embodiments, the recombinant expression vector is a viralconstruct, e.g., a recombinant adeno-associated virus construct (see,e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, arecombinant lentiviral construct, a recombinant retroviral construct,etc.

Suitable expression vectors include, but are not limited to, viralvectors (e.g. viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549,1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like.

Numerous suitable expression vectors are known to those of skill in theart, and many are commercially available. The following vectors areprovided by way of example; for eukaryotic host cells: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, anyother vector may be used so long as it is compatible with the host cell.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

In some embodiments, a nucleotide sequence encoding a DNA-targeting RNAand/or a site-directed modifying polypeptide is operably linked to acontrol element, e.g., a transcriptional control element, such as apromoter. The transcriptional control element may be functional ineither a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell(e.g., bacterial or archaeal cell). In some embodiments, a nucleotidesequence encoding a DNA-targeting RNA and/or a site-directed modifyingpolypeptide is operably linked to multiple control elements that allowexpression of the nucleotide sequence encoding a DNA-targeting RNAand/or a site-directed modifying polypeptide in both prokaryotic andeukaryotic cells.

Non-limiting examples of suitable eukaryotic promoters (promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, andmouse metallothionein-I. Selection of the appropriate vector andpromoter is well within the level of ordinary skill in the art. Theexpression vector may also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector may also include appropriate sequences for amplifying expression.The expression vector may also include nucleotide sequences encodingprotein tags (e.g., 6×His tag, hemagglutinin tag, green fluorescentprotein, etc.) that are fused to the site-directed modifyingpolypeptide, thus resulting in a chimeric polypeptide.

In some embodiments, a nucleotide sequence encoding a DNA-targeting RNAand/or a site-directed modifying polypeptide is operably linked to aninducible promoter. In some embodiments, a nucleotide sequence encodinga DNA-targeting RNA and/or a site-directed modifying polypeptide isoperably linked to a constitutive promoter.

Methods of introducing a nucleic acid into a host cell are known in theart, and any known method can be used to introduce a nucleic acid (e.g.,an expression construct) into a cell. Suitable methods include e.g.,viral or bacteriophage infection, transfection, conjugation, protoplastfusion, lipofection, electroporation, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro injection,nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., alAdv Drug Deliv Rev. 2012 Sep. 13. pii: S0169-409X(12)00283-9. doi:10.1016/j.addr.2012.09.023), and the like.

Chimeric Polypeptides

The present disclosure provides a chimeric site-directed modifyingpolypeptide. A subject chimeric site-directed modifying polypeptideinteracts with (e.g., binds to) a subject DNA-targeting RNA (describedabove). The DNA-targeting RNA guides the chimeric site-directedmodifying polypeptide to a target sequence within target DNA (e.g. achromosomal sequence or an extrachromosomal sequence, e.g. an episomalsequence, a minicircle sequence, a mitochondrial sequence, a chloroplastsequence, etc.). A subject chimeric site-directed modifying polypeptidemodifies target DNA (e.g., cleavage or methylation of target DNA) and/ora polypeptide associated with target DNA (e.g., methylation oracetylation of a histone tail).

A subject chimeric site-directed modifying polypeptide modifies targetDNA (e.g., cleavage or methylation of target DNA) and/or a polypeptideassociated with target DNA (e.g., methylation or acetylation of ahistone tail). A chimeric site-directed modifying polypeptide is alsoreferred to herein as a “chimeric site-directed polypeptide” or a“chimeric RNA binding site-directed modifying polypeptide.”

A subject chimeric site-directed modifying polypeptide comprises twoportions, an RNA-binding portion and an activity portion. A subjectchimeric site-directed modifying polypeptide comprises amino acidsequences that are derived from at least two different polypeptides. Asubject chimeric site-directed modifying polypeptide can comprisemodified and/or naturally-occurring polypeptide sequences (e.g., a firstamino acid sequence from a modified or unmodified Cas9/Csn1 protein; anda second amino acid sequence other than the Cas9/Csn1 protein).

RNA-Binding Portion

In some cases, the RNA-binding portion of a subject chimericsite-directed modifying polypeptide is a naturally-occurringpolypeptide. In other cases, the RNA-binding portion of a subjectchimeric site-directed modifying polypeptide is not anaturally-occurring molecule (modified, e.g., mutation, deletion,insertion). Naturally-occurring RNA-binding portions of interest arederived from site-directed modifying polypeptides known in the art. Forexample, SEQ ID NOs:1-256 and 795-1346 provide a non-limiting andnon-exhaustive list of naturally occurring Cas9/Csn1 endonucleases thatcan be used as site-directed modifying polypeptides. In some cases, theRNA-binding portion of a subject chimeric site-directed modifyingpolypeptide comprises an amino acid sequence having at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the RNA-binding portion of a polypeptide having anyof the amino acid sequences set forth as SEQ ID NOs:1-256 and 795-1346).

In some cases, the site-directed modifying polypeptide comprises anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, or 100%, amino acid sequence identity to amino acids 7-166 or731-1003 of the Cas9/Csn1 amino acid sequence depicted in FIG. 3A andFIG. 3B, or to the corresponding portions in any of the amino acidsequences set forth as SEQ ID NOs:1-256 and 795-1346.

Activity Portion

In addition to the RNA-binding portion, the chimeric site-directedmodifying polypeptide comprises an “activity portion.” In someembodiments, the activity portion of a subject chimeric site-directedmodifying polypeptide comprises the naturally-occurring activity portionof a site-directed modifying polypeptide (e.g., Cas9/Csn1 endonuclease).In other embodiments, the activity portion of a subject chimericsite-directed modifying polypeptide comprises a modified amino acidsequence (e.g., substitution, deletion, insertion) of anaturally-occurring activity portion of a site-directed modifyingpolypeptide. Naturally-occurring activity portions of interest arederived from site-directed modifying polypeptides known in the art. Forexample, SEQ ID NOs:1-256 and 795-1346 provide a non-limiting andnon-exhaustive list of naturally occurring Cas9/Csn1 endonucleases thatcan be used as site-directed modifying polypeptides. The activityportion of a subject chimeric site-directed modifying polypeptide isvariable and may comprise any heterologous polypeptide sequence that maybe useful in the methods disclosed herein.

In some embodiments, a subject chimeric site-directed modifyingpolypeptide comprises: (i) an RNA-binding portion that interacts with aDNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotidesequence that is complementary to a sequence in a target DNA; and (ii)an activity portion that exhibits site-directed enzymatic activity(e.g., activity for DNA methylation, activity for DNA cleavage, activityfor histone acetylation, activity for histone methylation, etc.),wherein the site of enzymatic activity is determined by theDNA-targeting RNA.

In other embodiments, a subject chimeric site-directed modifyingpolypeptide comprises: (i) an RNA-binding portion that interacts with aDNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotidesequence that is complementary to a sequence in a target DNA; and (ii)an activity portion that modulates transcription within the target DNA(e.g., to increase or decrease transcription), wherein the site ofmodulated transcription within the target DNA is determined by theDNA-targeting RNA.

In some cases, the activity portion of a subject chimeric site-directedmodifying polypeptide has enzymatic activity that modifies target DNA(e.g., nuclease activity, methyltransferase activity, demethylaseactivity, DNA repair activity, DNA damage activity, deaminationactivity, dismutase activity, alkylation activity, depurinationactivity, oxidation activity, pyrimidine dimer forming activity,integrase activity, transposase activity, recombinase activity,polymerase activity, ligase activity, helicase activity, photolyaseactivity or glycosylase activity).

In other cases, the activity portion of a subject chimeric site-directedmodifying polypeptide has enzymatic activity (e.g., methyltransferaseactivity, demethylase activity, acetyltransferase activity, deacetylaseactivity, kinase activity, phosphatase activity, ubiquitin ligaseactivity, deubiquitinating activity, adenylation activity, deadenylationactivity, SUMOylating activity, deSUMOylating activity, ribosylationactivity, deribosylation activity, myristoylation activity ordemyristoylation activity) that modifies a polypeptide associated withtarget DNA (e.g., a histone).

In some cases, the activity portion of a subject chimeric site-directedmodifying polypeptide exhibits enzymatic activity (described above). Inother cases, the activity portion of a subject chimeric site-directedmodifying polypeptide modulates transcription of the target DNA(described above). The activity portion of a subject chimericsite-directed modifying polypeptide is variable and may comprise anyheterologous polypeptide sequence that may be useful in the methodsdisclosed herein.

Exemplary Chimeric Site-Directed Modifying Polypeptides

In some embodiments, the activity portion of the chimeric site-directedmodifying polypeptide comprises a modified form of the Cas9/Csn1protein. In some instances, the modified form of the Cas9/Csn1 proteincomprises an amino acid change (e.g., deletion, insertion, orsubstitution) that reduces the naturally-occurring nuclease activity ofthe Cas9/Csn1 protein. For example, in some instances, the modified formof the Cas9/Csn1 protein has less than 50%, less than 40%, less than30%, less than 20%, less than 10%, less than 5%, or less than 1% of thenuclease activity of the corresponding wild-type Cas9/Csn1 polypeptide.In some cases, the modified form of the Cas9/Csn1 polypeptide has nosubstantial nuclease activity.

In some embodiments, the modified form of the Cas9/Csn1 polypeptide is aD10A (aspartate to alanine at amino acid position 10 of SEQ ID NO:8)mutation (or the corresponding mutation of any of the proteins presentedin SEQ ID NOs:1-256 and 795-1346) that can cleave the complementarystrand of the target DNA but has reduced ability to cleave thenon-complementary strand of the target DNA (see FIG. 11A-11B). In someembodiments, the modified form of the Cas9/Csn1 polypeptide is a H840A(histidine to alanine at amino acid position 840) mutation (or thecorresponding mutation of any of the proteins set forth as SEQ IDNOs:1-256 and 795-1346) that can cleave the non-complementary strand ofthe target DNA but has reduced ability to cleave the complementarystrand of the target DNA (see FIG. 11A-11B). In some embodiments, themodified form of the Cas9/Csn1 polypeptide harbors both the D10A and theH840A mutations (or the corresponding mutations of any of the proteinsset forth as SEQ ID NOs:1-256 and 795-1346) such that the polypeptidehas a reduced ability to cleave both the complementary and thenon-complementary strands of the target DNA. Other residues can bemutated to achieve the above effects (i.e. inactivate one or the othernuclease portions). As non-limiting examples, residues D10, G12, G17,E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 (or thecorresponding mutations of any of the proteins set forth as SEQ IDNOs:1-256 and 795-1346) can be altered (i.e., substituted) (see FIG.3A-3B, FIG. 5, FIG. 11A, and Table 1 for more information regarding theconservation of Cas9 amino acid residues). Also, mutations other thanalanine substitutions are suitable. For more information of important

TABLE 1 Table 1 lists 4 motifs that are present inCas9 sequences from various species (see alsoFIG. 3A-3B and FIG. 5). The amino acids listed hereare from the Cas9 from S. pyogenes (SEQ ID NO: 8). Motif # MotifAmino acids (residue #s) Highly conserved 1 RuvC-like IIGLDIGTNSVGWAVI (7-21) D10, G12, G17 (SEQ ID NO: 260) 2 RuvC-like IIIVIEMARE (759-766) E762 (SEQ ID NO: 261) 3 HNH-motifDVDHIVPQSFLKDDSIDNKVLTRSDKN H840, N854, N863 (837-863) (SEQ ID NO: 262)4 RuvC-like II HHAHDAYL (982-989) H982, H983, A984, (SEQ ID NO: 263)D986, A987

In some cases, the chimeric site-directed modifying polypeptidecomprises an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 99% or 100% amino acid sequence identity to amino acids7-166 or 731-1003 of the Cas9/Csn1 amino acid sequence depicted in FIG.3A and FIG. 3B, or to the corresponding portions in any of the aminoacid sequences set forth as SEQ ID NOs:1-256 and 795-1346. In somecases, the chimeric site-directed modifying polypeptide comprises 4motifs (as listed in Table 4 and depicted in FIG. 3A and FIG. 5), eachwith amino acid sequences having at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 99% or 100% amino acid sequence identity to each of the 4 motifslisted in Table 1 (SEQ ID NOs:260-263), or to the corresponding portionsin any of the amino acid sequences set forth as SEQ ID NOs:1-256 and795-1346. In some cases, the chimeric site-directed modifyingpolypeptide comprises amino acid sequences having at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 99% or 100% amino acid sequence identity to aminoacids 7-166 or 731-1003 of the Cas9/Csn1 amino acid sequence depicted inFIG. 3A and FIG. 3B, or to the corresponding portions in any of theamino acid sequences set forth as SEQ ID NOs:1-256 and 795-1346.

In some embodiments, the activity portion of the site-directed modifyingpolypeptide comprises a heterologous polypeptide that has DNA-modifyingactivity and/or transcription factor activity and/or DNA-associatedpolypeptide-modifying activity. In some cases, a heterologouspolypeptide replaces a portion of the Cas9/Csn1 polypeptide thatprovides nuclease activity. In other embodiments, a subjectsite-directed modifying polypeptide comprises both a portion of theCas9/Csn1 polypeptide that normally provides nuclease activity (and thatportion can be fully active or can instead be modified to have less than100% of the corresponding wild-type activity) and a heterologouspolypeptide. In other words, in some cases, a subject chimericsite-directed modifying polypeptide is a fusion polypeptide comprisingboth the portion of the Cas9/Csn1 polypeptide that normally providesnuclease activity and the heterologous polypeptide. In other cases, asubject chimeric site-directed modifying polypeptide is a fusionpolypeptide comprising a modified variant of the activity portion of theCas9/Csn1 polypeptide (e.g., amino acid change, deletion, insertion) anda heterologous polypeptide. In yet other cases, a subject chimericsite-directed modifying polypeptide is a fusion polypeptide comprising aheterologous polypeptide and the RNA-binding portion of anaturally-occurring or a modified site-directed modifying polypeptide.

For example, in a chimeric Cas9/Csn1 protein, a naturally-occurring (ormodified, e.g., mutation, deletion, insertion) bacterial Cas9/Csn1polypeptide may be fused to a heterologous polypeptide sequence (i.e. apolypeptide sequence from a protein other than Cas9/Csn1 or apolypeptide sequence from another organism). The heterologouspolypeptide sequence may exhibit an activity (e.g., enzymatic activity)that will also be exhibited by the chimeric Cas9/Csn1 protein (e.g.,methyltransferase activity, acetyltransferase activity, kinase activity,ubiquitinating activity, etc.). A heterologous nucleic acid sequence maybe linked to another nucleic acid sequence (e.g., by geneticengineering) to generate a chimeric nucleotide sequence encoding achimeric polypeptide. In some embodiments, a chimeric Cas9/Csn1polypeptide is generated by fusing a Cas9/Csn1 polypeptide (e.g., wildtype Cas9 or a Cas9 variant, e.g., a Cas9 with reduced or inactivatednuclease activity) with a heterologous sequence that provides forsubcellular localization (e.g., a nuclear localization signal (NLS) fortargeting to the nucleus; a mitochondrial localization signal fortargeting to the mitochondria; a chloroplast localization signal fortargeting to a chloroplast; an ER retention signal; and the like). Insome embodiments, the heterologous sequence can provide a tag for easeof tracking or purification (e.g., a fluorescent protein, e.g., greenfluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and thelike; a HIS tag, e.g., a 6×His tag; a hemagglutinin (HA) tag; a FLAGtag; a Myc tag; and the like). In some embodiments, the heterologoussequence can provide for increased or decreased stability. In someembodiments, the heterologous sequence can provide a binding domain(e.g., to provide the ability of a chimeric Cas9 polypeptide to bind toanother protein of interest, e.g., a DNA or histone modifying protein, atranscription factor or transcription repressor, a recruiting protein,etc.).

Examples of various additional suitable fusion partners (or fragmentsthereof) for a subject variant Cas9 site-directed polypeptide include,but are not limited to those listed in FIG. 54A-54C.

Nucleic Acid Encoding a Subject Chimeric Site-Directed ModifyingPolypeptide

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a subject chimeric site-directed modifyingpolypeptide. In some embodiments, the nucleic acid comprising anucleotide sequence encoding a subject chimeric site-directed modifyingpolypeptide is an expression vector, e.g., a recombinant expressionvector.

In some embodiments, a subject method involves contacting a target DNAor introducing into a cell (or a population of cells) one or morenucleic acids comprising a chimeric site-directed modifying polypeptide.Suitable nucleic acids comprising nucleotide sequences encoding achimeric site-directed modifying polypeptide include expression vectors,where an expression vector comprising a nucleotide sequence encoding achimeric site-directed modifying polypeptide is a “recombinantexpression vector.”

In some embodiments, the recombinant expression vector is a viralconstruct, e.g., a recombinant adeno-associated virus construct (see,e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, arecombinant lentiviral construct, etc.

Suitable expression vectors include, but are not limited to, viralvectors (e.g. viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549,1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like.

Numerous suitable expression vectors are known to those of skill in theart, and many are commercially available. The following vectors areprovided by way of example; for eukaryotic host cells: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, anyother vector may be used so long as it is compatible with the host cell.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

In some embodiments, a nucleotide sequence encoding a chimericsite-directed modifying polypeptide is operably linked to a controlelement, e.g., a transcriptional control element, such as a promoter.The transcriptional control element may be functional in either aeukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell (e.g.,bacterial or archaeal cell). In some embodiments, a nucleotide sequenceencoding a chimeric site-directed modifying polypeptide is operablylinked to multiple control elements that allow expression of thenucleotide sequence encoding a chimeric site-directed modifyingpolypeptide in both prokaryotic and eukaryotic cells.

Non-limiting examples of suitable eukaryotic promoters (promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, andmouse metallothionein-I. Selection of the appropriate vector andpromoter is well within the level of ordinary skill in the art. Theexpression vector may also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector may also include appropriate sequences for amplifying expression.The expression vector may also include nucleotide sequences encodingprotein tags (e.g., 6×His tag, hemagglutinin (HA) tag, a fluorescentprotein (e.g., a green fluorescent protein; a yellow fluorescentprotein, etc.), etc.) that are fused to the chimeric site-directedmodifying polypeptide.

In some embodiments, a nucleotide sequence encoding a chimericsite-directed modifying polypeptide is operably linked to an induciblepromoter (e.g., heat shock promoter, Tetracycline-regulated promoter,Steroid-regulated promoter, Metal-regulated promoter, estrogenreceptor-regulated promoter, etc.). In some embodiments, a nucleotidesequence encoding a chimeric site-directed modifying polypeptide isoperably linked to a spatially restricted and/or temporally restrictedpromoter (e.g., a tissue specific promoter, a cell type specificpromoter, etc.). In some embodiments, a nucleotide sequence encoding achimeric site-directed modifying polypeptide is operably linked to aconstitutive promoter.

Methods of introducing a nucleic acid into a host cell are known in theart, and any known method can be used to introduce a nucleic acid (e.g.,an expression construct) into a stem cell or progenitor cell. Suitablemethods include, include e.g., viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, calcium phosphate precipitation, polyethyleneimine(PEI)-mediated transfection, DEAE-dextran mediated transfection,liposome-mediated transfection, particle gun technology, calciumphosphate precipitation, direct micro injection, nanoparticle-mediatednucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev.2012 Sep. 13. pii: 50169-409X(12)00283-9. doi:10.1016/j.addr.2012.09.023), and the like.

Methods

The present disclosure provides methods for modifying a target DNAand/or a target DNA-associated polypeptide. Generally, a subject methodinvolves contacting a target DNA with a complex (a “targeting complex”),which complex comprises a DNA-targeting RNA and a site-directedmodifying polypeptide.

As discussed above, a subject DNA-targeting RNA and a subjectsite-directed modifying polypeptide form a complex. The DNA-targetingRNA provides target specificity to the complex by comprising anucleotide sequence that is complementary to a sequence of a target DNA.The site-directed modifying polypeptide of the complex provides thesite-specific activity. In some embodiments, a subject complex modifiesa target DNA, leading to, for example, DNA cleavage, DNA methylation,DNA damage, DNA repair, etc. In other embodiments, a subject complexmodifies a target polypeptide associated with target DNA (e.g., ahistone, a DNA-binding protein, etc.), leading to, for example, histonemethylation, histone acetylation, histone ubiquitination, and the like.The target DNA may be, for example, naked DNA in vitro, chromosomal DNAin cells in vitro, chromosomal DNA in cells in vivo, etc.

In some cases, the site-directed modifying polypeptide exhibits nucleaseactivity that cleaves target DNA at a target DNA sequence defined by theregion of complementarity between the DNA-targeting RNA and the targetDNA. In some cases, when the site-directed modifying polypeptide is aCas9 or Cas9 related polypeptide, site-specific cleavage of the targetDNA occurs at locations determined by both (i) base-pairingcomplementarity between the DNA-targeting RNA and the target DNA; and(ii) a short motif [referred to as the protospacer adjacent motif (PAM)]in the target DNA. In some embodiments (e.g., when Cas9 from S.pyogenes, or a closely related Cas9, is used (see SEQ ID NOs:1-256 and795-1346)), the PAM sequence of the non-complementary strand is5′-XGG-3′, where X is any DNA nucleotide and X is immediately 3′ of thetarget sequence of the non-complementary strand of the target DNA (seeFIG. 10A-10E). As such, the PAM sequence of the complementary strand is5′-CCY-3′, where Y is any DNA nucleotide and Y is immediately 5′ of thetarget sequence of the complementary strand of the target DNA (see FIG.10A-10E where the PAM of the non-complementary strand is 5′-GGG-3′ andthe PAM of the complementary strand is 5′-CCC-3′). In some suchembodiments, X and Y can be complementary and the X-Y base pair can beany basepair (e.g., X=C and Y=G; X=G and Y=C; X=A and Y=T, X=T and Y=A).

In some cases, different Cas9 proteins (i.e., Cas9 proteins from variousspecies) may be advantageous to use in the various provided methods inorder to capitalize on various enzymatic characteristics of thedifferent Cas9 proteins (e.g., for different PAM sequence preferences;for increased or decreased enzymatic activity; for an increased ordecreased level of cellular toxicity; to change the balance betweenNHEJ, homology-directed repair, single strand breaks, double strandbreaks, etc.). Cas9 proteins from various species (see SEQ ID NOs:1-256and 795-1346) may require different PAM sequences in the target DNA.Thus, for a particular Cas9 protein of choice, the PAM sequencerequirement may be different than the 5′-XGG-3′ sequence describedabove.

Many Cas9 orthologus from a wide variety of species have been identifiedherein and the proteins share only a few identical amino acids. Allidentified Cas9 orthologs have the same domain architecture with acentral HNH endonuclease domain and a split RuvC/RNaseH domain (See FIG.3A, FIG. 3B, FIG. 5, and Table 1). Cas9 proteins share 4 key motifs witha conserved architecture. Motifs 1, 2, and 4 are RuvC like motifs whilemotif 3 is an HNH-motif. In some cases, a suitable site-directedmodifying polypeptide comprises an amino acid sequence having 4 motifs,each of motifs 1-4 having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99% or 100% amino acid sequence identity to the motif s 1-4 of theCas9/Csn1 amino acid sequence depicted in FIG. 3A (SEQ ID NOs:260-263,respectively, as depicted in Table 1), or to the corresponding portionsin any of the amino acid sequences set forth in SEQ ID NOs:1-256 and795-1346 (see FIG. 5 for an alignment of motifs 1-4 from divergent Cas9sequences). In some cases, a suitable site-directed modifyingpolypeptide comprises an amino acid sequence having at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 99% or 100% amino acid sequence identity toamino acids 7-166 or 731-1003 of the Cas9/Csn1 amino acid sequencedepicted in FIG. 3A and FIG. 3B, or to the corresponding portions in anyof the amino acid sequences set forth as SEQ ID NOs:1-256 and 795-1346.Any Cas9 protein as defined above can be used as a site-directedmodifying polypeptide or as part of a chimeric site-directed modifyingpolypeptide of the subject methods.

The nuclease activity cleaves target DNA to produce double strandbreaks. These breaks are then repaired by the cell in one of two ways:non-homologous end joining, and homology-directed repair (FIG. 2). Innon-homologous end joining (NHEJ), the double-strand breaks are repairedby direct ligation of the break ends to one another. As such, no newnucleic acid material is inserted into the site, although some nucleicacid material may be lost, resulting in a deletion. In homology-directedrepair, a donor polynucleotide with homology to the cleaved target DNAsequence is used as a template for repair of the cleaved target DNAsequence, resulting in the transfer of genetic information from thedonor polynucleotide to the target DNA. As such, new nucleic acidmaterial may be inserted/copied into the site. In some cases, a targetDNA is contacted with a subject donor polynucleotide. In some cases, asubject donor polynucleotide is introduced into a subject cell. Themodifications of the target DNA due to NHEJ and/or homology-directedrepair lead to, for example, gene correction, gene replacement, genetagging, transgene insertion, nucleotide deletion, gene disruption, genemutation, etc.

Accordingly, cleavage of DNA by a site-directed modifying polypeptidemay be used to delete nucleic acid material from a target DNA sequence(e.g., to disrupt a gene that makes cells susceptible to infection (e.g.the CCR5 or CXCR4 gene, which makes T cells susceptible to HIVinfection), to remove disease-causing trinucleotide repeat sequences inneurons, to create gene knockouts and mutations as disease models inresearch, etc.) by cleaving the target DNA sequence and allowing thecell to repair the sequence in the absence of an exogenously provideddonor polynucleotide. Thus, the subject methods can be used to knock outa gene (resulting in complete lack of transcription or alteredtranscription) or to knock in genetic material into a locus of choice inthe target DNA.

Alternatively, if a DNA-targeting RNA and a site-directed modifyingpolypeptide are coadministered to cells with a donor polynucleotidesequence that includes at least a segment with homology to the targetDNA sequence, the subject methods may be used to add, i.e. insert orreplace, nucleic acid material to a target DNA sequence (e.g. to “knockin” a nucleic acid that encodes for a protein, an siRNA, an miRNA,etc.), to add a tag (e.g., 6×His, a fluorescent protein (e.g., a greenfluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin(HA), FLAG, etc.), to add a regulatory sequence to a gene (e.g.promoter, polyadenylation signal, internal ribosome entry sequence(IRES), 2A peptide, start codon, stop codon, splice signal, localizationsignal, etc.), to modify a nucleic acid sequence (e.g., introduce amutation), and the like. As such, a complex comprising a DNA-targetingRNA and a site-directed modifying polypeptide is useful in any in vitroor in vivo application in which it is desirable to modify DNA in asite-specific, i.e. “targeted”, way, for example gene knock-out, geneknock-in, gene editing, gene tagging, etc., as used in, for example,gene therapy, e.g. to treat a disease or as an antiviral,antipathogenic, or anticancer therapeutic, the production of geneticallymodified organisms in agriculture, the large scale production ofproteins by cells for therapeutic, diagnostic, or research purposes, theinduction of iPS cells, biological research, the targeting of genes ofpathogens for deletion or replacement, etc.

In some embodiments, the site-directed modifying polypeptide comprises amodified form of the Cas9/Csn1 protein. In some instances, the modifiedform of the Cas9/Csn1 protein comprises an amino acid change (e.g.,deletion, insertion, or substitution) that reduces thenaturally-occurring nuclease activity of the Cas9/Csn1 protein. Forexample, in some instances, the modified form of the Cas9/Csn1 proteinhas less than 50%, less than 40%, less than 30%, less than 20%, lessthan 10%, less than 5%, or less than 1% of the nuclease activity of thecorresponding wild-type Cas9/Csn1 polypeptide. In some cases, themodified form of the Cas9/Csn1 polypeptide has no substantial nucleaseactivity. When a subject site-directed modifying polypeptide is amodified form of the Cas9/Csn1 polypeptide that has no substantialnuclease activity, it can be referred to as “dCas9.”

In some embodiments, the modified form of the Cas9/Csn1 polypeptide is aD10A (aspartate to alanine at amino acid position 10 of SEQ ID NO:8)mutation (or the corresponding mutation of any of the proteins set forthas SEQ ID NOs:1-256 and 795-1346) that can cleave the complementarystrand of the target DNA but has reduced ability to cleave thenon-complementary strand of the target DNA (thus resulting in a singlestrand break (SSB) instead of a DSB; see FIG. 11A-11B). In someembodiments, the modified form of the Cas9/Csn1 polypeptide is a H840A(histidine to alanine at amino acid position 840 of SEQ ID NO:8)mutation (or the corresponding mutation of any of the proteins set forthas SEQ ID NOs:1-256 and 795-1346) that can cleave the non-complementarystrand of the target DNA but has reduced ability to cleave thecomplementary strand of the target DNA (thus resulting in a singlestrand break (SSB) instead of a DSB; see FIG. 11A-11B). The use of theD10A or H840A variant of Cas9 (or the corresponding mutations in any ofthe proteins set forth as SEQ ID NOs:1-256 and 795-1346) can alter theexpected biological outcome because the non-homologous end joining(NHEJ) is much more likely to occur when DSBs are present as opposed toSSBs. Thus, in some cases where one wishes to reduce the likelihood ofDSB (and therefore reduce the likelihood of NHEJ), a D10A or H840Avariant of Cas9 can be used. Other residues can be mutated to achievethe same effect (i.e. inactivate one or the other nuclease portions). Asnon-limiting examples, residues D10, G12, G17, E762, H840, N854, N863,H982, H983, A984, D986, and/or A987 (or the corresponding mutations ofany of the proteins set forth as SEQ ID NOs:1-256 and 795-1346) can bealtered (i.e., substituted) (see FIG. 3A-3B, FIG. 5, FIG. 11A, and Table1 for more information regarding the conservation of Cas9 amino acidresidues). Also, mutations other than alanine substitutions aresuitable. In some embodiments when a site-directed polypeptide (e.g.,site-directed modifying polypeptide) has reduced catalytic activity(e.g., when a Cas9 protein has a D10, G12, G17, E762, H840, N854, N863,H982, H983, A984, D986, and/or a A987 mutation, e.g., D10A, G12A, G17A,E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A), thepolypeptide can still bind to target DNA in a site-specific manner(because it is still guided to a target DNA sequence by a DNA-targetingRNA) as long as it retains the ability to interact with theDNA-targeting RNA.

In some embodiments, the modified form of the Cas9/Csn1 polypeptideharbors both the D10A and the H840A mutations (or the correspondingmutations of any of the proteins set forth as SEQ ID NOs:1-256 and795-1346) such that the polypeptide has a reduced ability to cleave boththe complementary and the non-complementary strands of the target DNA(i.e., the variant can have no substantial nuclease activity). Otherresidues can be mutated to achieve the same effect (i.e. inactivate oneor the other nuclease portions). As non-limiting examples, residues D10,G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987(or the corresponding mutations of any of the proteins set forth as SEQID NOs:1-256 and 795-1346) can be altered (i.e., substituted) (see FIG.3A-3B, FIG. 5, FIG. 11A, and Table 1 for more information regarding theconservation of Cas9 amino acid residues). Also, mutations other thanalanine substitutions are suitable.

In some embodiments, the site-directed modifying polypeptide comprises aheterologous sequence (e.g., a fusion). In some embodiments, aheterologous sequence can provide for subcellular localization of thesite-directed modifying polypeptide (e.g., a nuclear localization signal(NLS) for targeting to the nucleus; a mitochondrial localization signalfor targeting to the mitochondria; a chloroplast localization signal fortargeting to a chloroplast; a ER retention signal; and the like). Insome embodiments, a heterologous sequence can provide a tag for ease oftracking or purification (e.g., a fluorescent protein, e.g., greenfluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and thelike; a his tag, e.g., a 6×His tag; a hemagglutinin (HA) tag; a FLAGtag; a Myc tag; and the like). In some embodiments, the heterologoussequence can provide for increased or decreased stability.

In some embodiments, a subject site-directed modifying polypeptide canbe codon-optimized. This type of optimization is known in the art andentails the mutation of foreign-derived DNA to mimic the codonpreferences of the intended host organism or cell while encoding thesame protein. Thus, the codons are changed, but the encoded proteinremains unchanged. For example, if the intended target cell was a humancell, a human codon-optimized Cas9 (or variant, e.g., enzymaticallyinactive variant) would be a suitable site-directed modifyingpolypeptide (see SEQ ID NO:256 for an example). Any suitablesite-directed modifying polypeptide (e.g., any Cas9 such as any of thesequences set forth in SEQ ID NOs:1-256 and 795-1346) can be codonoptimized. As another non-limiting example, if the intended host cellwere a mouse cell, than a mouse codon-optimized Cas9 (or variant, e.g.,enzymatically inactive variant) would be a suitable site-directedmodifying polypeptide. While codon optimization is not required, it isacceptable and may be preferable in certain cases.

In some embodiments, a subject DNA-targeting RNA and a subjectsite-directed modifying polypeptide are used as an inducible system forshutting off gene expression in bacterial cells. In some cases, nucleicacids encoding an appropriate DNA-targeting RNA and/or an appropriatesite-directed polypeptide are incorporated into the chromosome of atarget cell and are under control of an inducible promoter. When theDNA-targeting RNA and/or the site-directed polypeptide are induced, thetarget DNA is cleaved (or otherwise modified) at the location ofinterest (e.g., a target gene on a separate plasmid), when both theDNA-targeting RNA and the site-directed modifying polypeptide arepresent and form a complex. As such, in some cases, bacterial expressionstrains are engineered to include nucleic acid sequences encoding anappropriate site-directed modifying polypeptide in the bacterial genomeand/or an appropriate DNA-targeting RNA on a plasmid (e.g., undercontrol of an inducible promoter), allowing experiments in which theexpression of any targeted gene (expressed from a separate plasmidintroduced into the strain) could be controlled by inducing expressionof the DNA-targeting RNA and the site-directed polypeptide.

In some cases, the site-directed modifying polypeptide has enzymaticactivity that modifies target DNA in ways other than introducing doublestrand breaks. Enzymatic activity of interest that may be used to modifytarget DNA (e.g., by fusing a heterologous polypeptide with enzymaticactivity to a site-directed modifying polypeptide, thereby generating achimeric site-directed modifying polypeptide) includes, but is notlimited methyltransferase activity, demethylase activity, DNA repairactivity, DNA damage activity, deamination activity, dismutase activity,alkylation activity, depurination activity, oxidation activity,pyrimidine dimer forming activity, integrase activity, transposaseactivity, recombinase activity, polymerase activity, ligase activity,helicase activity, photolyase activity or glycosylase activity).Methylation and demethylation is recognized in the art as an importantmode of epigenetic gene regulation while DNA damage and repair activityis essential for cell survival and for proper genome maintenance inresponse to environmental stresses.

As such, the methods herein find use in the epigenetic modification oftarget DNA and may be employed to control epigenetic modification oftarget DNA at any location in a target DNA by genetically engineeringthe desired complementary nucleic acid sequence into the DNA-targetingsegment of a DNA-targeting RNA. The methods herein also find use in theintentional and controlled damage of DNA at any desired location withinthe target DNA. The methods herein also find use in thesequence-specific and controlled repair of DNA at any desired locationwithin the target DNA. Methods to target DNA-modifying enzymaticactivities to specific locations in target DNA find use in both researchand clinical applications.

In some cases, the site-directed modifying polypeptide has activity thatmodulates the transcription of target DNA (e.g., in the case of achimeric site-directed modifying polypeptide, etc.). In some cases, achimeric site-directed modifying polypeptides comprising a heterologouspolypeptide that exhibits the ability to increase or decreasetranscription (e.g., transcriptional activator or transcriptionrepressor polypeptides) is used to increase or decrease thetranscription of target DNA at a specific location in a target DNA,which is guided by the DNA-targeting segment of the DNA-targeting RNA.Examples of source polypeptides for providing a chimeric site-directedmodifying polypeptide with transcription modulatory activity include,but are not limited to light-inducible transcription regulators, smallmolecule/drug-responsive transcription regulators, transcriptionfactors, transcription repressors, etc. In some cases, the subjectmethod is used to control the expression of a targeted coding-RNA(protein-encoding gene) and/or a targeted non-coding RNA (e.g., tRNA,rRNA, snoRNA, siRNA, miRNA, long ncRNA, etc.).

In some cases, the site-directed modifying polypeptide has enzymaticactivity that modifies a polypeptide associated with DNA (e.g. histone).In some embodiments, the enzymatic activity is methyltransferaseactivity, demethylase activity, acetyltransferase activity, deacetylaseactivity, kinase activity, phosphatase activity, ubiquitin ligaseactivity (i.e., ubiquitination activity), deubiquitinating activity,adenylation activity, deadenylation activity, SUMOylating activity,deSUMOylating activity, ribosylation activity, deribosylation activity,myristoylation activity, demyristoylation activity glycosylationactivity (e.g., from O-GlcNAc transferase) or deglycosylation activity.The enzymatic activities listed herein catalyze covalent modificationsto proteins. Such modifications are known in the art to alter thestability or activity of the target protein (e.g., phosphorylation dueto kinase activity can stimulate or silence protein activity dependingon the target protein). Of particular interest as protein targets arehistones. Histone proteins are known in the art to bind DNA and formcomplexes known as nucleosomes. Histones can be modified (e.g., bymethylation, acetylation, ubuitination, phosphorylation) to elicitstructural changes in the surrounding DNA, thus controlling theaccessibility of potentially large portions of DNA to interactingfactors such as transcription factors, polymerases and the like. Asingle histone can be modified in many different ways and in manydifferent combinations (e.g., trimethylation of lysine 27 of histone 3,H3K27, is associated with DNA regions of repressed transcription whiletrimethylation of lysine 4 of histone 3, H3K4, is associated with DNAregions of active transcription). Thus, a site-directed modifyingpolypeptide with histone-modifying activity finds use in the sitespecific control of DNA structure and can be used to alter the histonemodification pattern in a selected region of target DNA. Such methodsfind use in both research and clinical applications.

In some embodiments, multiple DNA-targeting RNAs are used simultaneouslyto simultaneously modify different locations on the same target DNA oron different target DNAs. In some embodiments, two or more DNA-targetingRNAs target the same gene or transcript or locus. In some embodiments,two or more DNA-targeting RNAs target different unrelated loci. In someembodiments, two or more DNA-targeting RNAs target different, butrelated loci.

In some cases, the site-directed modifying polypeptide is provideddirectly as a protein. As one non-limiting example, fungi (e.g., yeast)can be transformed with exogenous protein and/or nucleic acid usingspheroplast transformation (see Kawai et al., Bioeng Bugs. 2010November-December; 1(6):395-403: “Transformation of Saccharomycescerevisiae and other fungi: methods and possible underlying mechanism”;and Tanka et al., Nature. 2004 Mar. 18; 428(6980):323-8: “Conformationalvariations in an infectious protein determine prion strain differences”;both of which are herein incorporated by reference in their entirety).Thus, a site-directed modifying polypeptide (e.g., Cas9) can beincorporated into a spheroplast (with or without nucleic acid encoding aDNA-targeting RNA and with or without a donor polynucleotide) and thespheroplast can be used to introduce the content into a yeast cell. Asite-directed modifying polypeptide can be introduced into a cell(provided to the cell) by any convenient method; such methods are knownto those of ordinary skill in the art. As another non-limiting example,a site-directed modifying polypeptide can be injected directly into acell (e.g., with or without nucleic acid encoding a DNA-targeting RNAand with or without a donor polynucleotide), e.g., a cell of a zebrafishembryo, the pronucleus of a fertilized mouse oocyte, etc.

Target Cells of Interest

In some of the above applications, the subject methods may be employedto induce DNA cleavage, DNA modification, and/or transcriptionalmodulation in mitotic or post-mitotic cells in vivo and/or ex vivoand/or in vitro (e.g., to produce genetically modified cells that can bereintroduced into an individual). Because the DNA-targeting RNA providespecificity by hybridizing to target DNA, a mitotic and/or post-mitoticcell of interest in the disclosed methods may include a cell from anyorganism (e.g. a bacterial cell, an archaeal cell, a cell of asingle-cell eukaryotic organism, a plant cell, an algal cell, e.g.,Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsisgaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and thelike, a fungal cell (e.g., a yeast cell), an animal cell, a cell from aninvertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode,etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile,bird, mammal), a cell from a mammal, a cell from a rodent, a cell from ahuman, etc.).

Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonicstem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell; asomatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, amuscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitroor in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell,2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.). Cells may befrom established cell lines or they may be primary cells, where “primarycells”, “primary cell lines”, and “primary cultures” are usedinterchangeably herein to refer to cells and cells cultures that havebeen derived from a subject and allowed to grow in vitro for a limitednumber of passages, i.e. splittings, of the culture. For example,primary cultures are cultures that may have been passaged 0 times, 1time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enoughtimes go through the crisis stage. Typically, the primary cell lines ofthe present invention are maintained for fewer than 10 passages invitro. Target cells are in many embodiments unicellular organisms, orare grown in culture.

If the cells are primary cells, they may be harvest from an individualby any convenient method. For example, leukocytes may be convenientlyharvested by apheresis, leukocytapheresis, density gradient separation,etc., while cells from tissues such as skin, muscle, bone marrow,spleen, liver, pancreas, lung, intestine, stomach, etc. are mostconveniently harvested by biopsy. An appropriate solution may be usedfor dispersion or suspension of the harvested cells. Such solution willgenerally be a balanced salt solution, e.g. normal saline,phosphate-buffered saline (PBS), Hank's balanced salt solution, etc.,conveniently supplemented with fetal calf serum or other naturallyoccurring factors, in conjunction with an acceptable buffer at lowconcentration, generally from 5-25 mM. Convenient buffers include HEPES,phosphate buffers, lactate buffers, etc. The cells may be usedimmediately, or they may be stored, frozen, for long periods of time,being thawed and capable of being reused. In such cases, the cells willusually be frozen in 10% DMSO, 50% serum, 40% buffered medium, or someother such solution as is commonly used in the art to preserve cells atsuch freezing temperatures, and thawed in a manner as commonly known inthe art for thawing frozen cultured cells.

Nucleic Acids Encoding a Subject DNA-Targeting RNA and/or a SubjectSite-Directed Modifying Polypeptide

In some embodiments, a subject method involves contacting a target DNAor introducing into a cell (or a population of cells) one or morenucleic acids comprising nucleotide sequences encoding a DNA-targetingRNA and/or a site-directed modifying polypeptide and/or a donorpolynucleotide. Suitable nucleic acids comprising nucleotide sequencesencoding a DNA-targeting RNA and/or a site-directed modifyingpolypeptide include expression vectors, where an expression vectorcomprising a nucleotide sequence encoding a DNA-targeting RNA and/or asite-directed modifying polypeptide is a “recombinant expressionvector.”

In some embodiments, the recombinant expression vector is a viralconstruct, e.g., a recombinant adeno-associated virus construct (see,e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, arecombinant lentiviral construct, etc.

Suitable expression vectors include, but are not limited to, viralvectors (e.g. viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549,1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like.

Numerous suitable expression vectors are known to those of skill in theart, and many are commercially available. The following vectors areprovided by way of example; for eukaryotic host cells: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, anyother vector may be used so long as it is compatible with the host cell.

In some embodiments, a nucleotide sequence encoding a DNA-targeting RNAand/or a site-directed modifying polypeptide is operably linked to acontrol element, e.g., a transcriptional control element, such as apromoter. The transcriptional control element may be functional ineither a eukaryotic cell, e.g., a mammalian cell, or a prokaryotic cell(e.g., bacterial or archaeal cell). In some embodiments, a nucleotidesequence encoding a DNA-targeting RNA and/or a site-directed modifyingpolypeptide is operably linked to multiple control elements that allowexpression of the nucleotide sequence encoding a DNA-targeting RNAand/or a site-directed modifying polypeptide in both prokaryotic andeukaryotic cells.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(e.g., U6 promoter, H1 promoter, etc.; see above) (see e.g., Bitter etal. (1987) Methods in Enzymology, 153:516-544).

In some embodiments, a DNA-targeting RNA and/or a site-directedmodifying polypeptide can be provided as RNA. In such cases, theDNA-targeting RNA and/or the RNA encoding the site-directed modifyingpolypeptide can be produced by direct chemical synthesis or may betranscribed in vitro from a DNA encoding the DNA-targeting RNA. Methodsof synthesizing RNA from a DNA template are well known in the art. Insome cases, the DNA-targeting RNA and/or the RNA encoding thesite-directed modifying polypeptide will be synthesized in vitro usingan RNA polymerase enzyme (e.g., T7 polymerase, T3 polymerase, SP6polymerase, etc.). Once synthesized, the RNA may directly contact atarget DNA or may be introduced into a cell by any of the well-knowntechniques for introducing nucleic acids into cells (e.g.,microinjection, electroporation, transfection, etc).

Nucleotides encoding a DNA-targeting RNA (introduced either as DNA orRNA) and/or a site-directed modifying polypeptide (introduced as DNA orRNA) and/or a donor polynucleotide may be provided to the cells usingwell-developed transfection techniques; see, e.g. Angel and Yanik (2010)PLoS ONE 5(7): e11756, and the commercially available TransMessenger®reagents from Qiagen, Stemfect™ RNA Transfection Kit from Stemgent, andTransIT®-mRNA Transfection Kit from Mirus Bio LLC. See also Beumer etal. (2008) Efficient gene targeting in Drosophila by direct embryoinjection with zinc-finger nucleases. PNAS 105(50):19821-19826.Alternatively, nucleic acids encoding a DNA-targeting RNA and/or asite-directed modifying polypeptide and/or a chimeric site-directedmodifying polypeptide and/or a donor polynucleotide may be provided onDNA vectors. Many vectors, e.g. plasmids, cosmids, minicircles, phage,viruses, etc., useful for transferring nucleic acids into target cellsare available. The vectors comprising the nucleic acid(s) may bemaintained episomally, e.g. as plasmids, minicircle DNAs, viruses suchcytomegalovirus, adenovirus, etc., or they may be integrated into thetarget cell genome, through homologous recombination or randomintegration, e.g. retrovirus-derived vectors such as MMLV, HIV-1, ALV,etc.

Vectors may be provided directly to the subject cells. In other words,the cells are contacted with vectors comprising the nucleic acidencoding DNA-targeting RNA and/or a site-directed modifying polypeptideand/or a chimeric site-directed modifying polypeptide and/or a donorpolynucleotide such that the vectors are taken up by the cells. Methodsfor contacting cells with nucleic acid vectors that are plasmids,including electroporation, calcium chloride transfection,microinjection, and lipofection are well known in the art. For viralvector delivery, the cells are contacted with viral particles comprisingthe nucleic acid encoding a DNA-targeting RNA and/or a site-directedmodifying polypeptide and/or a chimeric site-directed modifyingpolypeptide and/or a donor polynucleotide. Retroviruses, for example,lentiviruses, are particularly suitable to the method of the invention.Commonly used retroviral vectors are “defective”, i.e. unable to produceviral proteins required for productive infection. Rather, replication ofthe vector requires growth in a packaging cell line. To generate viralparticles comprising nucleic acids of interest, the retroviral nucleicacids comprising the nucleic acid are packaged into viral capsids by apackaging cell line. Different packaging cell lines provide a differentenvelope protein (ecotropic, amphotropic or xenotropic) to beincorporated into the capsid, this envelope protein determining thespecificity of the viral particle for the cells (ecotropic for murineand rat; amphotropic for most mammalian cell types including human, dogand mouse; and xenotropic for most mammalian cell types except murinecells). The appropriate packaging cell line may be used to ensure thatthe cells are targeted by the packaged viral particles. Methods ofintroducing the retroviral vectors comprising the nucleic acid encodingthe reprogramming factors into packaging cell lines and of collectingthe viral particles that are generated by the packaging lines are wellknown in the art. Nucleic acids can also introduced by directmicro-injection (e.g., injection of RNA into a zebrafish embryo).

Vectors used for providing the nucleic acids encoding DNA-targeting RNAand/or a site-directed modifying polypeptide and/or a chimericsite-directed modifying polypeptide and/or a donor polynucleotide to thesubject cells will typically comprise suitable promoters for driving theexpression, that is, transcriptional activation, of the nucleic acid ofinterest. In other words, the nucleic acid of interest will be operablylinked to a promoter. This may include ubiquitously acting promoters,for example, the CMV-R-actin promoter, or inducible promoters, such aspromoters that are active in particular cell populations or that respondto the presence of drugs such as tetracycline. By transcriptionalactivation, it is intended that transcription will be increased abovebasal levels in the target cell by at least about 10 fold, by at leastabout 100 fold, more usually by at least about 1000 fold. In addition,vectors used for providing a DNA-targeting RNA and/or a site-directedmodifying polypeptide and/or a chimeric site-directed modifyingpolypeptide and/or a donor polynucleotide to the subject cells mayinclude nucleic acid sequences that encode for selectable markers in thetarget cells, so as to identify cells that have taken up theDNA-targeting RNA and/or a site-directed modifying polypeptide and/or achimeric site-directed modifying polypeptide and/or a donorpolynucleotide.

A subject DNA-targeting RNA and/or a site-directed modifying polypeptideand/or a chimeric site-directed modifying polypeptide may instead beused to contact DNA or introduced into cells as RNA. Methods ofintroducing RNA into cells are known in the art and may include, forexample, direct injection, transfection, or any other method used forthe introduction of DNA.

A subject site-directed modifying polypeptide may instead be provided tocells as a polypeptide. Such a polypeptide may optionally be fused to apolypeptide domain that increases solubility of the product. The domainmay be linked to the polypeptide through a defined protease cleavagesite, e.g. a TEV sequence, which is cleaved by TEV protease. The linkermay also include one or more flexible sequences, e.g. from 1 to 10glycine residues. In some embodiments, the cleavage of the fusionprotein is performed in a buffer that maintains solubility of theproduct, e.g. in the presence of from 0.5 to 2 M urea, in the presenceof polypeptides and/or polynucleotides that increase solubility, and thelike. Domains of interest include endosomolytic domains, e.g. influenzaHA domain; and other polypeptides that aid in production, e.g. IF2domain, GST domain, GRPE domain, and the like. The polypeptide may beformulated for improved stability. For example, the peptides may bePEGylated, where the polyethyleneoxy group provides for enhancedlifetime in the blood stream.

Additionally or alternatively, the subject site-directed modifyingpolypeptide may be fused to a polypeptide permeant domain to promoteuptake by the cell. A number of permeant domains are known in the artand may be used in the non-integrating polypeptides of the presentinvention, including peptides, peptidomimetics, and non-peptidecarriers. For example, a permeant peptide may be derived from the thirdalpha helix of Drosophila melanogaster transcription factorAntennapaedia, referred to as penetratin, which comprises the amino acidsequence RQIKIWFQNRRMKWKK (SEQ ID NO: 268). As another example, thepermeant peptide comprises the HIV-1 tat basic region amino acidsequence, which may include, for example, amino acids 49-57 ofnaturally-occurring tat protein. Other permeant domains includepolyarginine motifs, for example, the region of amino acids 34-56 ofHIV-1 rev protein, nona-arginine, octa-arginine, and the like. (See, forexample, Futaki et al. (2003) Curr Protein Pept Sci. 2003 April; 4(2):87-9 and 446; and Wender et al. (2000) Proc. Natl. Acad. Sci. U.S.A 2000Nov. 21; 97(24):13003-8; published U.S. Patent applications 20030220334;20030083256; 20030032593; and 20030022831, herein specificallyincorporated by reference for the teachings of translocation peptidesand peptoids). The nona-arginine (R9) sequence is one of the moreefficient PTDs that have been characterized (Wender et al. 2000; Uemuraet al. 2002). The site at which the fusion is made may be selected inorder to optimize the biological activity, secretion or bindingcharacteristics of the polypeptide. The optimal site will be determinedby routine experimentation.

A subject site-directed modifying polypeptide may be produced in vitroor by eukaryotic cells or by prokaryotic cells, and it may be furtherprocessed by unfolding, e.g. heat denaturation, DTT reduction, etc. andmay be further refolded, using methods known in the art.

Modifications of interest that do not alter primary sequence includechemical derivatization of polypeptides, e.g., acylation, acetylation,carboxylation, amidation, etc. Also included are modifications ofglycosylation, e.g. those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g. by exposing the polypeptide to enzymes whichaffect glycosylation, such as mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences that have phosphorylated amino acidresidues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

Also included in the subject invention are DNA-targeting RNAs andsite-directed modifying polypeptides that have been modified usingordinary molecular biological techniques and synthetic chemistry so asto improve their resistance to proteolytic degradation, to change thetarget sequence specificity, to optimize solubility properties, to alterprotein activity (e.g., transcription modulatory activity, enzymaticactivity, etc) or to render them more suitable as a therapeutic agent.Analogs of such polypeptides include those containing residues otherthan naturally occurring L-amino acids, e.g. D-amino acids ornon-naturally occurring synthetic amino acids. D-amino acids may besubstituted for some or all of the amino acid residues.

The site-directed modifying polypeptides may be prepared by in vitrosynthesis, using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example, automatedsynthesizers by Applied Biosystems, Inc., Beckman, etc. By usingsynthesizers, naturally occurring amino acids may be substituted withunnatural amino acids. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

The site-directed modifying polypeptides may also be isolated andpurified in accordance with conventional methods of recombinantsynthesis. A lysate may be prepared of the expression host and thelysate purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, or other purificationtechnique. For the most part, the compositions which are used willcomprise at least 20% by weight of the desired product, more usually atleast about 75% by weight, preferably at least about 95% by weight, andfor therapeutic purposes, usually at least about 99.5% by weight, inrelation to contaminants related to the method of preparation of theproduct and its purification. Usually, the percentages will be basedupon total protein.

To induce DNA cleavage and recombination, or any desired modification toa target DNA, or any desired modification to a polypeptide associatedwith target DNA, the DNA-targeting RNA and/or the site-directedmodifying polypeptide and/or the donor polynucleotide, whether they beintroduced as nucleic acids or polypeptides, are provided to the cellsfor about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours,8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other periodfrom about 30 minutes to about 24 hours, which may be repeated with afrequency of about every day to about every 4 days, e.g., every 1.5days, every 2 days, every 3 days, or any other frequency from aboutevery day to about every four days. The agent(s) may be provided to thesubject cells one or more times, e.g. one time, twice, three times, ormore than three times, and the cells allowed to incubate with theagent(s) for some amount of time following each contacting event e.g.16-24 hours, after which time the media is replaced with fresh media andthe cells are cultured further.

In cases in which two or more different targeting complexes are providedto the cell (e.g., two different DNA-targeting RNAs that arecomplementary to different sequences within the same or different targetDNA), the complexes may be provided simultaneously (e.g. as twopolypeptides and/or nucleic acids), or delivered simultaneously.Alternatively, they may be provided consecutively, e.g. the targetingcomplex being provided first, followed by the second targeting complex,etc. or vice versa.

Typically, an effective amount of the DNA-targeting RNA and/orsite-directed modifying polypeptide and/or donor polynucleotide isprovided to the target DNA or cells to induce cleavage. An effectiveamount of the DNA-targeting RNA and/or site-directed modifyingpolypeptide and/or donor polynucleotide is the amount to induce a 2-foldincrease or more in the amount of target modification observed betweentwo homologous sequences relative to a negative control, e.g. a cellcontacted with an empty vector or irrelevant polypeptide. That is tosay, an effective amount or dose of the DNA-targeting RNA and/orsite-directed modifying polypeptide and/or donor polynucleotide willinduce a 2-fold increase, a 3-fold increase, a 4-fold increase or morein the amount of target modification observed at a target DNA region, insome instances a 5-fold increase, a 6-fold increase or more, sometimes a7-fold or 8-fold increase or more in the amount of recombinationobserved, e.g. an increase of 10-fold, 50-fold, or 100-fold or more, insome instances, an increase of 200-fold, 500-fold, 700-fold, or1000-fold or more, e.g. a 5000-fold, or 10,000-fold increase in theamount of recombination observed. The amount of target modification maybe measured by any convenient method. For example, a silent reporterconstruct comprising complementary sequence to the targeting segment(targeting sequence) of the DNA-targeting RNA flanked by repeatsequences that, when recombined, will reconstitute a nucleic acidencoding an active reporter may be cotransfected into the cells, and theamount of reporter protein assessed after contact with the DNA-targetingRNA and/or site-directed modifying polypeptide and/or donorpolynucleotide, e.g. 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36hours, 48 hours, 72 hours or more after contact with the DNA-targetingRNA and/or site-directed modifying polypeptide and/or donorpolynucleotide. As another, more sensitivity assay, for example, theextent of recombination at a genomic DNA region of interest comprisingtarget DNA sequences may be assessed by PCR or Southern hybridization ofthe region after contact with a DNA-targeting RNA and/or site-directedmodifying polypeptide and/or donor polynucleotide, e.g. 2 hours, 4hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or moreafter contact with the DNA-targeting RNA and/or site-directed modifyingpolypeptide and/or donor polynucleotide.

Contacting the cells with a DNA-targeting RNA and/or site-directedmodifying polypeptide and/or donor polynucleotide may occur in anyculture media and under any culture conditions that promote the survivalof the cells. For example, cells may be suspended in any appropriatenutrient medium that is convenient, such as Iscove's modified DMEM orRPMI 1640, supplemented with fetal calf serum or heat inactivated goatserum (about 5-10%), L-glutamine, a thiol, particularly2-mercaptoethanol, and antibiotics, e.g. penicillin and streptomycin.The culture may contain growth factors to which the cells areresponsive. Growth factors, as defined herein, are molecules capable ofpromoting survival, growth and/or differentiation of cells, either inculture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors. Conditions that promote the survival of cellsare typically permissive of nonhomologous end joining andhomology-directed repair.

In applications in which it is desirable to insert a polynucleotidesequence into a target DNA sequence, a polynucleotide comprising a donorsequence to be inserted is also provided to the cell. By a “donorsequence” or “donor polynucleotide” it is meant a nucleic acid sequenceto be inserted at the cleavage site induced by a site-directed modifyingpolypeptide. The donor polynucleotide will contain sufficient homologyto a genomic sequence at the cleavage site, e.g. 70%, 80%, 85%, 90%,95%, or 100% homology with the nucleotide sequences flanking thecleavage site, e.g. within about 50 bases or less of the cleavage site,e.g. within about 30 bases, within about 15 bases, within about 10bases, within about 5 bases, or immediately flanking the cleavage site,to support homology-directed repair between it and the genomic sequenceto which it bears homology. Approximately 25, 50, 100, or 200nucleotides, or more than 200 nucleotides, of sequence homology betweena donor and a genomic sequence (or any integral value between 10 and 200nucleotides, or more) will support homology-directed repair. Donorsequences can be of any length, e.g. 10 nucleotides or more, 50nucleotides or more, 100 nucleotides or more, 250 nucleotides or more,500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides ormore, etc.

The donor sequence is typically not identical to the genomic sequencethat it replaces. Rather, the donor sequence may contain at least one ormore single base changes, insertions, deletions, inversions orrearrangements with respect to the genomic sequence, so long assufficient homology is present to support homology-directed repair. Insome embodiments, the donor sequence comprises a non-homologous sequenceflanked by two regions of homology, such that homology-directed repairbetween the target DNA region and the two flanking sequences results ininsertion of the non-homologous sequence at the target region. Donorsequences may also comprise a vector backbone containing sequences thatare not homologous to the DNA region of interest and that are notintended for insertion into the DNA region of interest. Generally, thehomologous region(s) of a donor sequence will have at least 50% sequenceidentity to a genomic sequence with which recombination is desired. Incertain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9%sequence identity is present. Any value between 1% and 100% sequenceidentity can be present, depending upon the length of the donorpolynucleotide.

The donor sequence may comprise certain sequence differences as comparedto the genomic sequence, e.g. restriction sites, nucleotidepolymorphisms, selectable markers (e.g., drug resistance genes,fluorescent proteins, enzymes etc.), etc., which may be used to assessfor successful insertion of the donor sequence at the cleavage site orin some cases may be used for other purposes (e.g., to signifyexpression at the targeted genomic locus). In some cases, if located ina coding region, such nucleotide sequence differences will not changethe amino acid sequence, or will make silent amino acid changes (i.e.,changes which do not affect the structure or function of the protein).Alternatively, these sequences differences may include flankingrecombination sequences such as FLPs, loxP sequences, or the like, thatcan be activated at a later time for removal of the marker sequence.

The donor sequence may be provided to the cell as single-stranded DNA,single-stranded RNA, double-stranded DNA, or double-stranded RNA. It maybe introduced into a cell in linear or circular form. If introduced inlinear form, the ends of the donor sequence may be protected (e.g., fromexonucleolytic degradation) by methods known to those of skill in theart. For example, one or more dideoxynucleotide residues are added tothe 3′ terminus of a linear molecule and/or self-complementaryoligonucleotides are ligated to one or both ends. See, for example,Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al.(1996) Science 272:886-889. Additional methods for protecting exogenouspolynucleotides from degradation include, but are not limited to,addition of terminal amino group(s) and the use of modifiedinternucleotide linkages such as, for example, phosphorothioates,phosphoramidates, and O-methyl ribose or deoxyribose residues. As analternative to protecting the termini of a linear donor sequence,additional lengths of sequence may be included outside of the regions ofhomology that can be degraded without impacting recombination. A donorsequence can be introduced into a cell as part of a vector moleculehaving additional sequences such as, for example, replication origins,promoters and genes encoding antibiotic resistance. Moreover, donorsequences can be introduced as naked nucleic acid, as nucleic acidcomplexed with an agent such as a liposome or poloxamer, or can bedelivered by viruses (e.g., adenovirus, AAV), as described above fornucleic acids encoding a DNA-targeting RNA and/or site-directedmodifying polypeptide and/or donor polynucleotide.

Following the methods described above, a DNA region of interest may becleaved and modified, i.e. “genetically modified”, ex vivo. In someembodiments, as when a selectable marker has been inserted into the DNAregion of interest, the population of cells may be enriched for thosecomprising the genetic modification by separating the geneticallymodified cells from the remaining population. Prior to enriching, the“genetically modified” cells may make up only about 1% or more (e.g., 2%or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8%or more, 9% or more, 10% or more, 15% or more, or 20% or more) of thecellular population. Separation of “genetically modified” cells may beachieved by any convenient separation technique appropriate for theselectable marker used. For example, if a fluorescent marker has beeninserted, cells may be separated by fluorescence activated cell sorting,whereas if a cell surface marker has been inserted, cells may beseparated from the heterogeneous population by affinity separationtechniques, e.g. magnetic separation, affinity chromatography, “panning”with an affinity reagent attached to a solid matrix, or other convenienttechnique. Techniques providing accurate separation include fluorescenceactivated cell sorters, which can have varying degrees ofsophistication, such as multiple color channels, low angle and obtuselight scattering detecting channels, impedance channels, etc. The cellsmay be selected against dead cells by employing dyes associated withdead cells (e.g. propidium iodide). Any technique may be employed whichis not unduly detrimental to the viability of the genetically modifiedcells. Cell compositions that are highly enriched for cells comprisingmodified DNA are achieved in this manner. By “highly enriched”, it ismeant that the genetically modified cells will be 70% or more, 75% ormore, 80% or more, 85% or more, 90% or more of the cell composition, forexample, about 95% or more, or 98% or more of the cell composition. Inother words, the composition may be a substantially pure composition ofgenetically modified cells.

Genetically modified cells produced by the methods described herein maybe used immediately. Alternatively, the cells may be frozen at liquidnitrogen temperatures and stored for long periods of time, being thawedand capable of being reused. In such cases, the cells will usually befrozen in 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium,or some other such solution as is commonly used in the art to preservecells at such freezing temperatures, and thawed in a manner as commonlyknown in the art for thawing frozen cultured cells.

The genetically modified cells may be cultured in vitro under variousculture conditions. The cells may be expanded in culture, i.e. grownunder conditions that promote their proliferation. Culture medium may beliquid or semi-solid, e.g. containing agar, methylcellulose, etc. Thecell population may be suspended in an appropriate nutrient medium, suchas Iscove's modified DMEM or RPMI 1640, normally supplemented with fetalcalf serum (about 5-10%), L-glutamine, a thiol, particularly2-mercaptoethanol, and antibiotics, e.g. penicillin and streptomycin.The culture may contain growth factors to which the regulatory T cellsare responsive. Growth factors, as defined herein, are molecules capableof promoting survival, growth and/or differentiation of cells, either inculture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors.

Cells that have been genetically modified in this way may betransplanted to a subject for purposes such as gene therapy, e.g. totreat a disease or as an antiviral, antipathogenic, or anticancertherapeutic, for the production of genetically modified organisms inagriculture, or for biological research. The subject may be a neonate, ajuvenile, or an adult. Of particular interest are mammalian subjects.Mammalian species that may be treated with the present methods includecanines and felines; equines; bovines; ovines; etc. and primates,particularly humans. Animal models, particularly small mammals (e.g.mouse, rat, guinea pig, hamster, lagomorpha (e.g., rabbit), etc.) may beused for experimental investigations.

Cells may be provided to the subject alone or with a suitable substrateor matrix, e.g. to support their growth and/or organization in thetissue to which they are being transplanted. Usually, at least 1×103cells will be administered, for example 5×103 cells, 1×104 cells, 5×104cells, 1×105 cells, 1×106 cells or more. The cells may be introduced tothe subject via any of the following routes: parenteral, subcutaneous,intravenous, intracranial, intraspinal, intraocular, or into spinalfluid. The cells may be introduced by injection, catheter, or the like.Examples of methods for local delivery, that is, delivery to the site ofinjury, include, e.g. through an Ommaya reservoir, e.g. for intrathecaldelivery (see e.g. U.S. Pat. Nos. 5,222,982 and 5,385,582, incorporatedherein by reference); by bolus injection, e.g. by a syringe, e.g. into ajoint; by continuous infusion, e.g. by cannulation, e.g. with convection(see e.g. US Application No. 20070254842, incorporated here byreference); or by implanting a device upon which the cells have beenreversably affixed (see e.g. US Application Nos. 20080081064 and20090196903, incorporated herein by reference). Cells may also beintroduced into an embryo (e.g., a blastocyst) for the purpose ofgenerating a transgenic animal (e.g., a transgenic mouse).

The number of administrations of treatment to a subject may vary.Introducing the genetically modified cells into the subject may be aone-time event; but in certain situations, such treatment may elicitimprovement for a limited period of time and require an on-going seriesof repeated treatments. In other situations, multiple administrations ofthe genetically modified cells may be required before an effect isobserved. The exact protocols depend upon the disease or condition, thestage of the disease and parameters of the individual subject beingtreated.

In other aspects of the invention, the DNA-targeting RNA and/orsite-directed modifying polypeptide and/or donor polynucleotide areemployed to modify cellular DNA in vivo, again for purposes such as genetherapy, e.g. to treat a disease or as an antiviral, antipathogenic, oranticancer therapeutic, for the production of genetically modifiedorganisms in agriculture, or for biological research. In these in vivoembodiments, a DNA-targeting RNA and/or site-directed modifyingpolypeptide and/or donor polynucleotide are administered directly to theindividual. A DNA-targeting RNA and/or site-directed modifyingpolypeptide and/or donor polynucleotide may be administered by any of anumber of well-known methods in the art for the administration ofpeptides, small molecules and nucleic acids to a subject. ADNA-targeting RNA and/or site-directed modifying polypeptide and/ordonor polynucleotide can be incorporated into a variety of formulations.More particularly, a DNA-targeting RNA and/or site-directed modifyingpolypeptide and/or donor polynucleotide of the present invention can beformulated into pharmaceutical compositions by combination withappropriate pharmaceutically acceptable carriers or diluents.

Pharmaceutical preparations are compositions that include one or more aDNA-targeting RNA and/or site-directed modifying polypeptide and/ordonor polynucleotide present in a pharmaceutically acceptable vehicle.“Pharmaceutically acceptable vehicles” may be vehicles approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inmammals, such as humans. The term “vehicle” refers to a diluent,adjuvant, excipient, or carrier with which a compound of the inventionis formulated for administration to a mammal. Such pharmaceuticalvehicles can be lipids, e.g. liposomes, e.g. liposome dendrimers;liquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like, saline; gum acacia, gelatin, starch paste,talc, keratin, colloidal silica, urea, and the like. In addition,auxiliary, stabilizing, thickening, lubricating and coloring agents maybe used. Pharmaceutical compositions may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants, gels, microspheres, and aerosols. As such,administration of the a DNA-targeting RNA and/or site-directed modifyingpolypeptide and/or donor polynucleotide can be achieved in various ways,including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intratracheal, intraocular, etc.,administration. The active agent may be systemic after administration ormay be localized by the use of regional administration, intramuraladministration, or use of an implant that acts to retain the active doseat the site of implantation. The active agent may be formulated forimmediate activity or it may be formulated for sustained release.

For some conditions, particularly central nervous system conditions, itmay be necessary to formulate agents to cross the blood-brain barrier(BBB). One strategy for drug delivery through the blood-brain barrier(BBB) entails disruption of the BBB, either by osmotic means such asmannitol or leukotrienes, or biochemically by the use of vasoactivesubstances such as bradykinin. The potential for using BBB opening totarget specific agents to brain tumors is also an option. A BBBdisrupting agent can be co-administered with the therapeuticcompositions of the invention when the compositions are administered byintravascular injection. Other strategies to go through the BBB mayentail the use of endogenous transport systems, including Caveolin-1mediated transcytosis, carrier-mediated transporters such as glucose andamino acid carriers, receptor-mediated transcytosis for insulin ortransferrin, and active efflux transporters such as p-glycoprotein.Active transport moieties may also be conjugated to the therapeuticcompounds for use in the invention to facilitate transport across theendothelial wall of the blood vessel. Alternatively, drug delivery oftherapeutics agents behind the BBB may be by local delivery, for exampleby intrathecal delivery, e.g. through an Ommaya reservoir (see e.g. U.S.Pat. Nos. 5,222,982 and 5,385,582, incorporated herein by reference); bybolus injection, e.g. by a syringe, e.g. intravitreally orintracranially; by continuous infusion, e.g. by cannulation, e.g. withconvection (see e.g. US Application No. 20070254842, incorporated hereby reference); or by implanting a device upon which the agent has beenreversably affixed (see e.g. US Application Nos. 20080081064 and20090196903, incorporated herein by reference).

Typically, an effective amount of a DNA-targeting RNA and/orsite-directed modifying polypeptide and/or donor polynucleotide areprovided. As discussed above with regard to ex vivo methods, aneffective amount or effective dose of a DNA-targeting RNA and/orsite-directed modifying polypeptide and/or donor polynucleotide in vivois the amount to induce a 2 fold increase or more in the amount ofrecombination observed between two homologous sequences relative to anegative control, e.g. a cell contacted with an empty vector orirrelevant polypeptide. The amount of recombination may be measured byany convenient method, e.g. as described above and known in the art. Thecalculation of the effective amount or effective dose of a DNA-targetingRNA and/or site-directed modifying polypeptide and/or donorpolynucleotide to be administered is within the skill of one of ordinaryskill in the art, and will be routine to those persons skilled in theart. The final amount to be administered will be dependent upon theroute of administration and upon the nature of the disorder or conditionthat is to be treated.

The effective amount given to a particular patient will depend on avariety of factors, several of which will differ from patient topatient. A competent clinician will be able to determine an effectiveamount of a therapeutic agent to administer to a patient to halt orreverse the progression the disease condition as required. UtilizingLD50 animal data, and other information available for the agent, aclinician can determine the maximum safe dose for an individual,depending on the route of administration. For instance, an intravenouslyadministered dose may be more than an intrathecally administered dose,given the greater body of fluid into which the therapeutic compositionis being administered. Similarly, compositions which are rapidly clearedfrom the body may be administered at higher doses, or in repeated doses,in order to maintain a therapeutic concentration. Utilizing ordinaryskill, the competent clinician will be able to optimize the dosage of aparticular therapeutic in the course of routine clinical trials.

For inclusion in a medicament, a DNA-targeting RNA and/or site-directedmodifying polypeptide and/or donor polynucleotide may be obtained from asuitable commercial source. As a general proposition, the totalpharmaceutically effective amount of the a DNA-targeting RNA and/orsite-directed modifying polypeptide and/or donor polynucleotideadministered parenterally per dose will be in a range that can bemeasured by a dose response curve.

Therapies based on a DNA-targeting RNA and/or site-directed modifyingpolypeptide and/or donor polynucleotides, i.e. preparations of aDNA-targeting RNA and/or site-directed modifying polypeptide and/ordonor polynucleotide to be used for therapeutic administration, must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 μm membranes). Therapeutic compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle. The therapies based on a DNA-targetingRNA and/or site-directed modifying polypeptide and/or donorpolynucleotide may be stored in unit or multi-dose containers, forexample, sealed ampules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-mL vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous solution of compound, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized compound using bacteriostaticWater-for-Injection.

Pharmaceutical compositions can include, depending on the formulationdesired, pharmaceutically-acceptable, non-toxic carriers of diluents,which are defined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, buffered water, physiologicalsaline, PBS, Ringer's solution, dextrose solution, and Hank's solution.In addition, the pharmaceutical composition or formulation can includeother carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenicstabilizers, excipients and the like. The compositions can also includeadditional substances to approximate physiological conditions, such aspH adjusting and buffering agents, toxicity adjusting agents, wettingagents and detergents.

The composition can also include any of a variety of stabilizing agents,such as an antioxidant for example. When the pharmaceutical compositionincludes a polypeptide, the polypeptide can be complexed with variouswell-known compounds that enhance the in vivo stability of thepolypeptide, or otherwise enhance its pharmacological properties (e.g.,increase the half-life of the polypeptide, reduce its toxicity, enhancesolubility or uptake). Examples of such modifications or complexingagents include sulfate, gluconate, citrate and phosphate. The nucleicacids or polypeptides of a composition can also be complexed withmolecules that enhance their in vivo attributes. Such molecules include,for example, carbohydrates, polyamines, amino acids, other peptides,ions (e.g., sodium, potassium, calcium, magnesium, manganese), andlipids.

Further guidance regarding formulations that are suitable for varioustypes of administration can be found in Remington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).For a brief review of methods for drug delivery, see, Langer, Science249:1527-1533 (1990).

The pharmaceutical compositions can be administered for prophylacticand/or therapeutic treatments. Toxicity and therapeutic efficacy of theactive ingredient can be determined according to standard pharmaceuticalprocedures in cell cultures and/or experimental animals, including, forexample, determining the LD50 (the dose lethal to 50% of the population)and the ED50 (the dose therapeutically effective in 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index and it can be expressed as the ratio LD50/ED50.Therapies that exhibit large therapeutic indices are preferred.

The data obtained from cell culture and/or animal studies can be used informulating a range of dosages for humans. The dosage of the activeingredient typically lines within a range of circulating concentrationsthat include the ED50 with low toxicity. The dosage can vary within thisrange depending upon the dosage form employed and the route ofadministration utilized.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

The effective amount of a therapeutic composition to be given to aparticular patient will depend on a variety of factors, several of whichwill differ from patient to patient. A competent clinician will be ableto determine an effective amount of a therapeutic agent to administer toa patient to halt or reverse the progression the disease condition asrequired. Utilizing LD50 animal data, and other information availablefor the agent, a clinician can determine the maximum safe dose for anindividual, depending on the route of administration. For instance, anintravenously administered dose may be more than an intrathecallyadministered dose, given the greater body of fluid into which thetherapeutic composition is being administered. Similarly, compositionswhich are rapidly cleared from the body may be administered at higherdoses, or in repeated doses, in order to maintain a therapeuticconcentration. Utilizing ordinary skill, the competent clinician will beable to optimize the dosage of a particular therapeutic in the course ofroutine clinical trials.

Genetically Modified Host Cells

The present disclosure provides genetically modified host cells,including isolated genetically modified host cells, where a subjectgenetically modified host cell comprises (has been genetically modifiedwith: 1) an exogenous DNA-targeting RNA; 2) an exogenous nucleic acidcomprising a nucleotide sequence encoding a DNA-targeting RNA; 3) anexogenous site-directed modifying polypeptide (e.g., a naturallyoccurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimericCas9; etc.); 4) an exogenous nucleic acid comprising a nucleotidesequence encoding a site-directed modifying polypeptide; or 5) anycombination of the above. A subject genetically modified cell isgenerated by genetically modifying a host cell with, for example: 1) anexogenous DNA-targeting RNA; 2) an exogenous nucleic acid comprising anucleotide sequence encoding a DNA-targeting RNA; 3) an exogenoussite-directed modifying polypeptide; 4) an exogenous nucleic acidcomprising a nucleotide sequence encoding a site-directed modifyingpolypeptide; or 5) any combination of the above).

All cells suitable to be a target cell are also suitable to be agenetically modified host cell. For example, a genetically modified hostcells of interest can be a cell from any organism (e.g. a bacterialcell, an archaeal cell, a cell of a single-cell eukaryotic organism, aplant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonasreinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassumpatens C. Agardh, and the like, a fungal cell (e.g., a yeast cell), ananimal cell, a cell from an invertebrate animal (e.g. fruit fly,cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal(e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal(e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, anon-human primate, a human, etc.), etc.

In some embodiments, a genetically modified host cell has beengenetically modified with an exogenous nucleic acid comprising anucleotide sequence encoding a site-directed modifying polypeptide(e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant,Cas9; a chimeric Cas9; etc.). The DNA of a genetically modified hostcell can be targeted for modification by introducing into the cell aDNA-targeting RNA (or a DNA encoding a DNA-targeting RNA, whichdetermines the genomic location/sequence to be modified) and optionallya donor nucleic acid. In some embodiments, the nucleotide sequenceencoding a site-directed modifying polypeptide is operably linked to aninducible promoter (e.g., heat shock promoter, Tetracycline-regulatedpromoter, Steroid-regulated promoter, Metal-regulated promoter, estrogenreceptor-regulated promoter, etc.). In some embodiments, the nucleotidesequence encoding a site-directed modifying polypeptide is operablylinked to a spatially restricted and/or temporally restricted promoter(e.g., a tissue specific promoter, a cell type specific promoter, etc.).In some embodiments, the nucleotide sequence encoding a site-directedmodifying polypeptide is operably linked to a constitutive promoter.

In some embodiments, a subject genetically modified host cell is invitro. In some embodiments, a subject genetically modified host cell isin vivo. In some embodiments, a subject genetically modified host cellis a prokaryotic cell or is derived from a prokaryotic cell. In someembodiments, a subject genetically modified host cell is a bacterialcell or is derived from a bacterial cell. In some embodiments, a subjectgenetically modified host cell is an archaeal cell or is derived from anarchaeal cell. In some embodiments, a subject genetically modified hostcell is a eukaryotic cell or is derived from a eukaryotic cell. In someembodiments, a subject genetically modified host cell is a plant cell oris derived from a plant cell. In some embodiments, a subject geneticallymodified host cell is an animal cell or is derived from an animal cell.In some embodiments, a subject genetically modified host cell is aninvertebrate cell or is derived from an invertebrate cell. In someembodiments, a subject genetically modified host cell is a vertebratecell or is derived from a vertebrate cell. In some embodiments, asubject genetically modified host cell is a mammalian cell or is derivedfrom a mammalian cell. In some embodiments, a subject geneticallymodified host cell is a rodent cell or is derived from a rodent cell. Insome embodiments, a subject genetically modified host cell is a humancell or is derived from a human cell.

The present disclosure further provides progeny of a subject geneticallymodified cell, where the progeny can comprise the same exogenous nucleicacid or polypeptide as the subject genetically modified cell from whichit was derived. The present disclosure further provides a compositioncomprising a subject genetically modified host cell.

Genetically Modified Stem Cells and Genetically Modified ProgenitorCells

In some embodiments, a subject genetically modified host cell is agenetically modified stem cell or progenitor cell. Suitable host cellsinclude, e.g., stem cells (adult stem cells, embryonic stem cells, iPScells, etc.) and progenitor cells (e.g., cardiac progenitor cells,neural progenitor cells, etc.). Suitable host cells include mammalianstem cells and progenitor cells, including, e.g., rodent stem cells,rodent progenitor cells, human stem cells, human progenitor cells, etc.Suitable host cells include in vitro host cells, e.g., isolated hostcells.

In some embodiments, a subject genetically modified host cell comprisesan exogenous DNA-targeting RNA nucleic acid. In some embodiments, asubject genetically modified host cell comprises an exogenous nucleicacid comprising a nucleotide sequence encoding a DNA-targeting RNA. Insome embodiments, a subject genetically modified host cell comprises anexogenous site-directed modifying polypeptide (e.g., a naturallyoccurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimericCas9; etc.). In some embodiments, a subject genetically modified hostcell comprises an exogenous nucleic acid comprising a nucleotidesequence encoding a site-directed modifying polypeptide. In someembodiments, a subject genetically modified host cell comprisesexogenous nucleic acid comprising a nucleotide sequence encoding 1) aDNA-targeting RNA and 2) a site-directed modifying polypeptide.

In some cases, the site-directed modifying polypeptide comprises anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, or 100%, amino acid sequence identity to amino acids 7-166 or731-1003 of the Cas9/Csn1 amino acid sequence depicted in FIG. 3A andFIG. 3B, or to the corresponding portions in any of the amino acidsequences set forth as SEQ ID NOs:1-256 and 795-1346.

Compositions

The present invention provides a composition comprising a subjectDNA-targeting RNA and/or a site-directed modifying polypeptide. In somecases, the site-directed modifying polypeptide is a subject chimericpolypeptide. A subject composition is useful for carrying out a methodof the present disclosure, e.g., a method for site-specific modificationof a target DNA; a method for site-specific modification of apolypeptide associated with a target DNA; etc.

Compositions Comprising a DNA-Targeting RNA

The present invention provides a composition comprising a subjectDNA-targeting RNA. The composition can comprise, in addition to theDNA-targeting RNA, one or more of: a salt, e.g., NaCl, MgCl₂, KCl,MgSO₄, etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES), MES sodium salt,3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a nuclease inhibitor; and the like. For example, in somecases, a subject composition comprises a subject DNA-targeting RNA and abuffer for stabilizing nucleic acids.

In some embodiments, a DNA-targeting RNA present in a subjectcomposition is pure, e.g., at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or more than 99% pure, where “% purity” meansthat DNA-targeting RNA is the recited percent free from othermacromolecules, or contaminants that may be present during theproduction of the DNA-targeting RNA.

Compositions Comprising a Subject Chimeric Polypeptide

The present invention provides a composition a subject chimericpolypeptide. The composition can comprise, in addition to theDNA-targeting RNA, one or more of: a salt, e.g., NaCl, MgCl₂, KCl,MgSO₄, etc.; a buffering agent, e.g., a Tris buffer, HEPES, MES, MESsodium salt, MOPS, TAPS, etc.; a solubilizing agent; a detergent, e.g.,a non-ionic detergent such as Tween-20, etc.; a protease inhibitor; areducing agent (e.g., dithiothreitol); and the like.

In some embodiments, a subject chimeric polypeptide present in a subjectcomposition is pure, e.g., at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or more than 99% pure, where “% purity” meansthat the site-directed modifying polypeptide is the recited percent freefrom other proteins, other macromolecules, or contaminants that may bepresent during the production of the chimeric polypeptide.

Compositions Comprising a DNA-Targeting RNA and a Site-DirectedModifying Polypeptide

The present invention provides a composition comprising: (i) aDNA-targeting RNA or a DNA polynucleotide encoding the same; and ii) asite-directed modifying polypeptide, or a polynucleotide encoding thesame. In some cases, the site-directed modifying polypeptide is asubject chimeric site-directed modifying polypeptide. In other cases,the site-directed modifying polypeptide is a naturally-occurringsite-directed modifying polypeptide. In some instances, thesite-directed modifying polypeptide exhibits enzymatic activity thatmodifies a target DNA. In other cases, the site-directed modifyingpolypeptide exhibits enzymatic activity that modifies a polypeptide thatis associated with a target DNA. In still other cases, the site-directedmodifying polypeptide modulates transcription of the target DNA.

The present invention provides a composition comprising: (i) aDNA-targeting RNA, as described above, or a DNA polynucleotide encodingthe same, the DNA-targeting RNA comprising: (a) a first segmentcomprising a nucleotide sequence that is complementary to a sequence ina target DNA; and (b) a second segment that interacts with asite-directed modifying polypeptide; and (ii) the site-directedmodifying polypeptide, or a polynucleotide encoding the same, thesite-directed modifying polypeptide comprising: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that exhibits site-directed enzymatic activity, wherein the siteof enzymatic activity is determined by the DNA-targeting RNA.

In some instances, a subject composition comprises: a compositioncomprising: (i) a subject DNA-targeting RNA, the DNA-targeting RNAcomprising: (a) a first segment comprising a nucleotide sequence that iscomplementary to a sequence in a target DNA; and (b) a second segmentthat interacts with a site-directed modifying polypeptide; and (ii) thesite-directed modifying polypeptide, the site-directed modifyingpolypeptide comprising: (a) an RNA-binding portion that interacts withthe DNA-targeting RNA; and (b) an activity portion that exhibitssite-directed enzymatic activity, wherein the site of enzymatic activityis determined by the DNA-targeting RNA.

In other embodiments, a subject composition comprises: (i) apolynucleotide encoding a subject DNA-targeting RNA, the DNA-targetingRNA comprising: (a) a first segment comprising a nucleotide sequencethat is complementary to a sequence in a target DNA; and (b) a secondsegment that interacts with a site-directed modifying polypeptide; and(ii) a polynucleotide encoding the site-directed modifying polypeptide,the site-directed modifying polypeptide comprising: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that exhibits site-directed enzymatic activity, wherein the siteof enzymatic activity is determined by the DNA-targeting RNA.

In some embodiments, a subject composition includes both RNA moleculesof a double-molecule DNA-targeting RNA. As such, in some embodiments, asubject composition includes an activator-RNA that comprises aduplex-forming segment that is complementary to the duplex-formingsegment of a targeter-RNA (see FIG. 1A). The duplex-forming segments ofthe activator-RNA and the targeter-RNA hybridize to form the dsRNAduplex of the protein-binding segment of the DNA-targeting RNA. Thetargeter-RNA further provides the DNA-targeting segment (singlestranded) of the DNA-targeting RNA and therefore targets theDNA-targeting RNA to a specific sequence within the target DNA. As onenon-limiting example, the duplex-forming segment of the activator-RNAcomprises a nucleotide sequence that has at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 98%,or 100% identity with the sequence 5′-UAGCAAGUUAAAAU-3′ (SEQ ID NO:562).As another non-limiting example, the duplex-forming segment of thetargeter-RNA comprises a nucleotide sequence that has at least about70%, at least about 80%, at least about 90%, at least about 95%, atleast about 98%, or 100% identity with the sequence 5′-GUUUUAGAGCUA-3′(SEQ ID NO:679).

The present disclosure provides a composition comprising: (i) aDNA-targeting RNA, or a DNA polynucleotide encoding the same, theDNA-targeting RNA comprising: (a) a first segment comprising anucleotide sequence that is complementary to a sequence in a target DNA;and (b) a second segment that interacts with a site-directed modifyingpolypeptide; and (ii) the site-directed modifying polypeptide, or apolynucleotide encoding the same, the site-directed modifyingpolypeptide comprising: (a) an RNA-binding portion that interacts withthe DNA-targeting RNA; and (b) an activity portion that modulatestranscription within the target DNA, wherein the site of modulatedtranscription within the target DNA is determined by the DNA-targetingRNA.

For example, in some cases, a subject composition comprises: (i) aDNA-targeting RNA, the DNA-targeting RNA comprising: (a) a first segmentcomprising a nucleotide sequence that is complementary to a sequence ina target DNA; and (b) a second segment that interacts with asite-directed modifying polypeptide; and (ii) the site-directedmodifying polypeptide, the site-directed modifying polypeptidecomprising: (a) an RNA-binding portion that interacts with theDNA-targeting RNA; and (b) an activity portion that modulatestranscription within the target DNA, wherein the site of modulatedtranscription within the target DNA is determined by the DNA-targetingRNA.

As another example, in some cases, a subject composition comprises: (i)a DNA polynucleotide encoding a DNA-targeting RNA, the DNA-targeting RNAcomprising: (a) a first segment comprising a nucleotide sequence that iscomplementary to a sequence in a target DNA; and (b) a second segmentthat interacts with a site-directed modifying polypeptide; and (ii) apolynucleotide encoding the site-directed modifying polypeptide, thesite-directed modifying polypeptide comprising: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that modulates transcription within the target DNA, wherein thesite of modulated transcription within the target DNA is determined bythe DNA-targeting RNA.

A subject composition can comprise, in addition to i) a subjectDNA-targeting RNA, or a DNA polynucleotide encoding the same; and ii) asite-directed modifying polypeptide, or a polynucleotide encoding thesame, one or more of: a salt, e.g., NaCl, MgCl₂, KCl, MgSO₄, etc.; abuffering agent, e.g., a Tris buffer, HEPES, MES, MES sodium salt, MOPS,TAPS, etc.; a solubilizing agent; a detergent, e.g., a non-ionicdetergent such as Tween-20, etc.; a protease inhibitor; a reducing agent(e.g., dithiothreitol); and the like.

In some cases, the components of the composition are individually pure,e.g., each of the components is at least about 75%, at least about 80%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or at least 99%, pure. In some cases, the individualcomponents of a subject composition are pure before being added to thecomposition.

For example, in some embodiments, a site-directed modifying polypeptidepresent in a subject composition is pure, e.g., at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or more than 99% pure,where “% purity” means that the site-directed modifying polypeptide isthe recited percent free from other proteins (e.g., proteins other thanthe site-directed modifying polypeptide), other macromolecules, orcontaminants that may be present during the production of thesite-directed modifying polypeptide.

Kits

The present disclosure provides kits for carrying out a subject method.A subject kit can include one or more of: a site-directed modifyingpolypeptide; a nucleic acid comprising a nucleotide encoding asite-directed modifying polypeptide; a DNA-targeting RNA; a nucleic acidcomprising a nucleotide sequence encoding a DNA-targeting RNA; anactivator-RNA; a nucleic acid comprising a nucleotide sequence encodingan activator-RNA; a targeter-RNA; and a nucleic acid comprising anucleotide sequence encoding a targeter-RNA. A site-directed modifyingpolypeptide; a nucleic acid comprising a nucleotide encoding asite-directed modifying polypeptide; a DNA-targeting RNA; a nucleic acidcomprising a nucleotide sequence encoding a DNA-targeting RNA; anactivator-RNA; a nucleic acid comprising a nucleotide sequence encodingan activator-RNA; a targeter-RNA; and a nucleic acid comprising anucleotide sequence encoding a targeter-RNA, are described in detailabove. A kit may comprise a complex that comprises two or more of: asite-directed modifying polypeptide; a nucleic acid comprising anucleotide encoding a site-directed modifying polypeptide; aDNA-targeting RNA; a nucleic acid comprising a nucleotide sequenceencoding a DNA-targeting RNA; an activator-RNA; a nucleic acidcomprising a nucleotide sequence encoding an activator-RNA; atargeter-RNA; and a nucleic acid comprising a nucleotide sequenceencoding a targeter-RNA.

In some embodiments, a subject kit comprises a site-directed modifyingpolypeptide, or a polynucleotide encoding the same. In some embodiments,the site-directed modifying polypeptide comprises: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that modulates transcription within the target DNA, wherein thesite of modulated transcription within the target DNA is determined bythe DNA-targeting RNA. In some cases, the activity portion of thesite-directed modifying polypeptide exhibits reduced or inactivatednuclease activity. In some cases, the site-directed modifyingpolypeptide is a chimeric site-directed modifying polypeptide.

In some embodiments, a subject kit comprises: a site-directed modifyingpolypeptide, or a polynucleotide encoding the same, and a reagent forreconstituting and/or diluting the site-directed modifying polypeptide.In other embodiments, a subject kit comprises a nucleic acid (e.g., DNA,RNA) comprising a nucleotide encoding a site-directed modifyingpolypeptide. In some embodiments, a subject kit comprises: a nucleicacid (e.g., DNA, RNA) comprising a nucleotide encoding a site-directedmodifying polypeptide; and a reagent for reconstituting and/or dilutingthe site-directed modifying polypeptide.

A subject kit comprising a site-directed modifying polypeptide, or apolynucleotide encoding the same, can further include one or moreadditional reagents, where such additional reagents can be selectedfrom: a buffer for introducing the site-directed modifying polypeptideinto a cell; a wash buffer; a control reagent; a control expressionvector or RNA polynucleotide; a reagent for in vitro production of thesite-directed modifying polypeptide from DNA, and the like. In somecases, the site-directed modifying polypeptide included in a subject kitis a chimeric site-directed modifying polypeptide, as described above.

In some embodiments, a subject kit comprises a DNA-targeting RNA, or aDNA polynucleotide encoding the same, the DNA-targeting RNA comprising:(a) a first segment comprising a nucleotide sequence that iscomplementary to a sequence in a target DNA; and (b) a second segmentthat interacts with a site-directed modifying polypeptide. In someembodiments, the DNA-targeting RNA further comprises a third segment (asdescribed above). In some embodiments, a subject kit comprises: (i) aDNA-targeting RNA, or a DNA polynucleotide encoding the same, theDNA-targeting RNA comprising: (a) a first segment comprising anucleotide sequence that is complementary to a sequence in a target DNA;and (b) a second segment that interacts with a site-directed modifyingpolypeptide; and (ii) a site-directed modifying polypeptide, or apolynucleotide encoding the same, the site-directed modifyingpolypeptide comprising: (a) an RNA-binding portion that interacts withthe DNA-targeting RNA; and (b) an activity portion that exhibitssite-directed enzymatic activity, wherein the site of enzymatic activityis determined by the DNA-targeting RNA. In some embodiments, theactivity portion of the site-directed modifying polypeptide does notexhibit enzymatic activity (comprises an inactivated nuclease, e.g., viamutation). In some cases, the kit comprises a DNA-targeting RNA and asite-directed modifying polypeptide. In other cases, the kit comprises:(i) a nucleic acid comprising a nucleotide sequence encoding aDNA-targeting RNA; and (ii) a nucleic acid comprising a nucleotidesequence encoding site-directed modifying polypeptide.

As another example, a subject kit can include: (i) a DNA-targeting RNA,or a DNA polynucleotide encoding the same, comprising: (a) a firstsegment comprising a nucleotide sequence that is complementary to asequence in a target DNA; and (b) a second segment that interacts with asite-directed modifying polypeptide; and (ii) the site-directedmodifying polypeptide, or a polynucleotide encoding the same,comprising: (a) an RNA-binding portion that interacts with theDNA-targeting RNA; and (b) an activity portion that that modulatestranscription within the target DNA, wherein the site of modulatedtranscription within the target DNA is determined by the DNA-targetingRNA In some cases, the kit comprises: (i) a DNA-targeting RNA; and asite-directed modifying polypeptide. In other cases, the kit comprises:(i) a nucleic acid comprising a nucleotide sequence encoding aDNA-targeting RNA; and (ii) a nucleic acid comprising a nucleotidesequence encoding site-directed modifying polypeptide.

The present disclosure provides a kit comprising: (1) a recombinantexpression vector comprising (i) a nucleotide sequence encoding aDNA-targeting RNA, wherein the DNA-targeting RNA comprises: (a) a firstsegment comprising a nucleotide sequence that is complementary to asequence in a target DNA; and (b) a second segment that interacts with asite-directed modifying polypeptide; and (ii) a nucleotide sequenceencoding the site-directed modifying polypeptide, wherein thesite-directed modifying polypeptide comprises: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that exhibits site-directed enzymatic activity, wherein the siteof enzymatic activity is determined by the DNA-targeting RNA; and (2) areagent for reconstitution and/or dilution of the expression vector.

The present disclosure provides a kit comprising: (1) a recombinantexpression vector comprising: (i) a nucleotide sequence encoding aDNA-targeting RNA, wherein the DNA-targeting RNA comprises: (a) a firstsegment comprising a nucleotide sequence that is complementary to asequence in a target DNA; and (b) a second segment that interacts with asite-directed modifying polypeptide; and (ii) a nucleotide sequenceencoding the site-directed modifying polypeptide, wherein thesite-directed modifying polypeptide comprises: (a) an RNA-bindingportion that interacts with the DNA-targeting RNA; and (b) an activityportion that modulates transcription within the target DNA, wherein thesite of modulated transcription within the target DNA is determined bythe DNA-targeting RNA; and (2) a reagent for reconstitution and/ordilution of the recombinant expression vector.

The present disclosure provides a kit comprising: (1) a recombinantexpression vector comprising a nucleic acid comprising a nucleotidesequence that encodes a DNA targeting RNA comprising: (i) a firstsegment comprising a nucleotide sequence that is complementary to asequence in a target DNA; and (ii) a second segment that interacts witha site-directed modifying polypeptide; and (2) a reagent forreconstitution and/or dilution of the recombinant expression vector. Insome embodiments of this kit, the kit comprises: a recombinantexpression vector comprising a nucleotide sequence that encodes asite-directed modifying polypeptide, wherein the site-directed modifyingpolypeptide comprises: (a) an RNA-binding portion that interacts withthe DNA-targeting RNA; and (b) an activity portion that exhibitssite-directed enzymatic activity, wherein the site of enzymatic activityis determined by the DNA-targeting RNA. In other embodiments of thiskit, the kit comprises: a recombinant expression vector comprising anucleotide sequence that encodes a site-directed modifying polypeptide,wherein the site-directed modifying polypeptide comprises: (a) anRNA-binding portion that interacts with the DNA-targeting RNA; and (b)an activity portion that modulates transcription within the target DNA,wherein the site of modulated transcription within the target DNA isdetermined by the DNA-targeting RNA.

In some embodiments of any of the above kits, the kit comprises anactivator-RNA or a targeter-RNA. In some embodiments of any of the abovekits, the kit comprises a single-molecule DNA-targeting RNA. In someembodiments of any of the above kits, the kit comprises two or moredouble-molecule or single-molecule DNA-targeting RNAs. In someembodiments of any of the above kits, a DNA-targeting RNA (e.g.,including two or more DNA-targeting RNAs) can be provided as an array(e.g., an array of RNA molecules, an array of DNA molecules encoding theDNA-targeting RNA(s), etc.). Such kits can be useful, for example, foruse in conjunction with the above described genetically modified hostcells that comprise a subject site-directed modifying polypeptide. Insome embodiments of any of the above kits, the kit further comprises adonor polynucleotide to effect the desired genetic modification.Components of a subject kit can be in separate containers; or can becombined in a single container.

Any of the above-described kits can further include one or moreadditional reagents, where such additional reagents can be selectedfrom: a dilution buffer; a reconstitution solution; a wash buffer; acontrol reagent; a control expression vector or RNA polynucleotide; areagent for in vitro production of the site-directed modifyingpolypeptide from DNA, and the like.

In addition to above-mentioned components, a subject kit can furtherinclude instructions for using the components of the kit to practice thesubject methods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

Non-Human Genetically Modified Organisms

In some embodiments, a genetically modified host cell has beengenetically modified with an exogenous nucleic acid comprising anucleotide sequence encoding a site-directed modifying polypeptide(e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant,Cas9; a chimeric Cas9; etc.). If such a cell is a eukaryotic single-cellorganism, then the modified cell can be considered a geneticallymodified organism. In some embodiments, subject non-human geneticallymodified organism is a Cas9 transgenic multicellular organism.

In some embodiments, a subject genetically modified non-human host cell(e.g., a cell that has been genetically modified with an exogenousnucleic acid comprising a nucleotide sequence encoding a site-directedmodifying polypeptide, e.g., a naturally occurring Cas9; a modified,i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) can generate asubject genetically modified non-human organism (e.g., a mouse, a fish,a frog, a fly, a worm, etc.). For example, if the genetically modifiedhost cell is a pluripotent stem cell (i.e., PSC) or a germ cell (e.g.,sperm, oocyte, etc.), an entire genetically modified organism can bederived from the genetically modified host cell. In some embodiments,the genetically modified host cell is a pluripotent stem cell (e.g.,ESC, iPSC, pluripotent plant stem cell, etc.) or a germ cell (e.g.,sperm cell, oocyte, etc.), either in vivo or in vitro, that can giverise to a genetically modified organism. In some embodiments thegenetically modified host cell is a vertebrate PSC (e.g., ESC, iPSC,etc.) and is used to generate a genetically modified organism (e.g. byinjecting a PSC into a blastocyst to produce a chimeric/mosaic animal,which could then be mated to generate non-chimeric/non-mosaicgenetically modified organisms; grafting in the case of plants; etc.).Any convenient method/protocol for producing a genetically modifiedorganism, including the methods described herein, is suitable forproducing a genetically modified host cell comprising an exogenousnucleic acid comprising a nucleotide sequence encoding a site-directedmodifying polypeptide (e.g., a naturally occurring Cas9; a modified,i.e., mutated or variant, Cas9; a chimeric Cas9; etc.). Methods ofproducing genetically modified organisms are known in the art. Forexample, see Cho et al., Curr Protoc Cell Biol. 2009 March; Chapter 19:Unit 19.11: Generation of transgenic mice; Gama et al., Brain StructFunct. 2010 March; 214(2-3):91-109. Epub 2009 Nov. 25: Animaltransgenesis: an overview; Husaini et al., GM Crops. 2011 June-December;2(3):150-62. Epub 2011 Jun. 1: Approaches for gene targeting andtargeted gene expression in plants.

In some embodiments, a genetically modified organism comprises a targetcell for methods of the invention, and thus can be considered a sourcefor target cells. For example, if a genetically modified cell comprisingan exogenous nucleic acid comprising a nucleotide sequence encoding asite-directed modifying polypeptide (e.g., a naturally occurring Cas9; amodified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) is usedto generate a genetically modified organism, then the cells of thegenetically modified organism comprise the exogenous nucleic acidcomprising a nucleotide sequence encoding a site-directed modifyingpolypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutatedor variant, Cas9; a chimeric Cas9; etc.). In some such embodiments, theDNA of a cell or cells of the genetically modified organism can betargeted for modification by introducing into the cell or cells aDNA-targeting RNA (or a DNA encoding a DNA-targeting RNA) and optionallya donor nucleic acid. For example, the introduction of a DNA-targetingRNA (or a DNA encoding a DNA-targeting RNA) into a subset of cells(e.g., brain cells, intestinal cells, kidney cells, lung cells, bloodcells, etc.) of the genetically modified organism can target the DNA ofsuch cells for modification, the genomic location of which will dependon the DNA-targeting sequence of the introduced DNA-targeting RNA.

In some embodiments, a genetically modified organism is a source oftarget cells for methods of the invention. For example, a geneticallymodified organism comprising cells that are genetically modified with anexogenous nucleic acid comprising a nucleotide sequence encoding asite-directed modifying polypeptide (e.g., a naturally occurring Cas9; amodified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) canprovide a source of genetically modified cells, for example PSCs (e.g.,ESCs, iPSCs, sperm, oocytes, etc.), neurons, progenitor cells,cardiomyocytes, etc.

In some embodiments, a genetically modified cell is a PSC comprising anexogenous nucleic acid comprising a nucleotide sequence encoding asite-directed modifying polypeptide (e.g., a naturally occurring Cas9; amodified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.). Assuch, the PSC can be a target cell such that the DNA of the PSC can betargeted for modification by introducing into the PSC a DNA-targetingRNA (or a DNA encoding a DNA-targeting RNA) and optionally a donornucleic acid, and the genomic location of the modification will dependon the DNA-targeting sequence of the introduced DNA-targeting RNA. Thus,in some embodiments, the methods described herein can be used to modifythe DNA (e.g., delete and/or replace any desired genomic location) ofPSCs derived from a subject genetically modified organism. Such modifiedPSCs can then be used to generate organisms having both (i) an exogenousnucleic acid comprising a nucleotide sequence encoding a site-directedmodifying polypeptide (e.g., a naturally occurring Cas9; a modified,i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) and (ii) a DNAmodification that was introduced into the PSC.

An exogenous nucleic acid comprising a nucleotide sequence encoding asite-directed modifying polypeptide (e.g., a naturally occurring Cas9; amodified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) can beunder the control of (i.e., operably linked to) an unknown promoter(e.g., when the nucleic acid randomly integrates into a host cellgenome) or can be under the control of (i.e., operably linked to) aknown promoter. Suitable known promoters can be any known promoter andinclude constitutively active promoters (e.g., CMV promoter), induciblepromoters (e.g., heat shock promoter, Tetracycline-regulated promoter,Steroid-regulated promoter, Metal-regulated promoter, estrogenreceptor-regulated promoter, etc.), spatially restricted and/ortemporally restricted promoters (e.g., a tissue specific promoter, acell type specific promoter, etc.), etc.

A subject genetically modified organism (e.g. an organism whose cellscomprise a nucleotide sequence encoding a site-directed modifyingpolypeptide, e.g., a naturally occurring Cas9; a modified, i.e., mutatedor variant, Cas9; a chimeric Cas9; etc.) can be any organism includingfor example, a plant; algae; an invertebrate (e.g., a cnidarian, anechinoderm, a worm, a fly, etc.); a vertebrate (e.g., a fish (e.g.,zebrafish, puffer fish, gold fish, etc.), an amphibian (e.g.,salamander, frog, etc.), a reptile, a bird, a mammal, etc.); an ungulate(e.g., a goat, a pig, a sheep, a cow, etc.); a rodent (e.g., a mouse, arat, a hamster, a guinea pig); a lagomorpha (e.g., a rabbit); etc.

In some cases, the site-directed modifying polypeptide comprises anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, or 100%, amino acid sequence identity to amino acids 7-166 or731-1003 of the Cas9/Csn1 amino acid sequence depicted in FIG. 3A andFIG. 3B, or to the corresponding portions in any of the amino acidsequences set forth as SEQ ID NOs:1-256 and 795-1346.

Transgenic Non-Human Animals

As described above, in some embodiments, a subject nucleic acid (e.g., anucleotide sequence encoding a site-directed modifying polypeptide,e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant,Cas9; a chimeric Cas9; etc.) or a subject recombinant expression vectoris used as a transgene to generate a transgenic animal that produces asite-directed modifying polypeptide. Thus, the present invention furtherprovides a transgenic non-human animal, which animal comprises atransgene comprising a subject nucleic acid comprising a nucleotidesequence encoding a site-directed modifying polypeptide, e.g., anaturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; achimeric Cas9; etc., as described above. In some embodiments, the genomeof the transgenic non-human animal comprises a subject nucleotidesequence encoding a site-directed modifying polypeptide. In someembodiments, the transgenic non-human animal is homozygous for thegenetic modification. In some embodiments, the transgenic non-humananimal is heterozygous for the genetic modification. In someembodiments, the transgenic non-human animal is a vertebrate, forexample, a fish (e.g., zebra fish, gold fish, puffer fish, cave fish,etc.), an amphibian (frog, salamander, etc.), a bird (e.g., chicken,turkey, etc.), a reptile (e.g., snake, lizard, etc.), a mammal (e.g., anungulate, e.g., a pig, a cow, a goat, a sheep, etc.; a lagomorph (e.g.,a rabbit); a rodent (e.g., a rat, a mouse); a non-human primate; etc.),etc.

An exogenous nucleic acid comprising a nucleotide sequence encoding asite-directed modifying polypeptide (e.g., a naturally occurring Cas9; amodified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) can beunder the control of (i.e., operably linked to) an unknown promoter(e.g., when the nucleic acid randomly integrates into a host cellgenome) or can be under the control of (i.e., operably linked to) aknown promoter. Suitable known promoters can be any known promoter andinclude constitutively active promoters (e.g., CMV promoter), induciblepromoters (e.g., heat shock promoter, Tetracycline-regulated promoter,Steroid-regulated promoter, Metal-regulated promoter, estrogenreceptor-regulated promoter, etc.), spatially restricted and/ortemporally restricted promoters (e.g., a tissue specific promoter, acell type specific promoter, etc.), etc.

In some cases, the site-directed modifying polypeptide comprises anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, or 100%, amino acid sequence identity to amino acids 7-166 or731-1003 of the Cas9/Csn1 amino acid sequence depicted in FIG. 3A andFIG. 3B, or to the corresponding portions in any of the amino acidsequences set forth as SEQ ID NOs:1-256 and 795-1346.

Transgenic Plants

As described above, in some embodiments, a subject nucleic acid (e.g., anucleotide sequence encoding a site-directed modifying polypeptide,e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant,Cas9; a chimeric Cas9; etc.) or a subject recombinant expression vectoris used as a transgene to generate a transgenic plant that produces asite-directed modifying polypeptide. Thus, the present invention furtherprovides a transgenic plant, which plant comprises a transgenecomprising a subject nucleic acid comprising a nucleotide sequenceencoding site-directed modifying polypeptide, e.g., a naturallyoccurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimericCas9; etc., as described above. In some embodiments, the genome of thetransgenic plant comprises a subject nucleic acid. In some embodiments,the transgenic plant is homozygous for the genetic modification. In someembodiments, the transgenic plant is heterozygous for the geneticmodification.

Methods of introducing exogenous nucleic acids into plant cells are wellknown in the art. Such plant cells are considered “transformed,” asdefined above. Suitable methods include viral infection (such as doublestranded DNA viruses), transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calcium phosphateprecipitation, direct microinjection, silicon carbide whiskerstechnology, Agrobacterium-mediated transformation and the like. Thechoice of method is generally dependent on the type of cell beingtransformed and the circumstances under which the transformation istaking place (i.e. in vitro, ex vivo, or in vivo).

Transformation methods based upon the soil bacterium Agrobacteriumtumefaciens are particularly useful for introducing an exogenous nucleicacid molecule into a vascular plant. The wild type form of Agrobacteriumcontains a Ti (tumor-inducing) plasmid that directs production oftumorigenic crown gall growth on host plants. Transfer of thetumor-inducing T-DNA region of the Ti plasmid to a plant genome requiresthe Ti plasmid-encoded virulence genes as well as T-DNA borders, whichare a set of direct DNA repeats that delineate the region to betransferred. An Agrobacterium-based vector is a modified form of a Tiplasmid, in which the tumor inducing functions are replaced by thenucleic acid sequence of interest to be introduced into the plant host.

Agrobacterium-mediated transformation generally employs cointegratevectors or binary vector systems, in which the components of the Tiplasmid are divided between a helper vector, which resides permanentlyin the Agrobacterium host and carries the virulence genes, and a shuttlevector, which contains the gene of interest bounded by T-DNA sequences.A variety of binary vectors are well known in the art and arecommercially available, for example, from Clontech (Palo Alto, Calif.).Methods of coculturing Agrobacterium with cultured plant cells orwounded tissue such as leaf tissue, root explants, hypocotyledons, stempieces or tubers, for example, also are well known in the art. See,e.g., Glick and Thompson, (eds.), Methods in Plant Molecular Biology andBiotechnology, Boca Raton, Fla.: CRC Press (1993).

Microprojectile-mediated transformation also can be used to produce asubject transgenic plant. This method, first described by Klein et al.(Nature 327:70-73 (1987)), relies on microprojectiles such as gold ortungsten that are coated with the desired nucleic acid molecule byprecipitation with calcium chloride, spermidine or polyethylene glycol.The microprojectile particles are accelerated at high speed into anangiosperm tissue using a device such as the BIOLISTIC PD-1000 (Biorad;Hercules Calif.).

A subject nucleic acid may be introduced into a plant in a manner suchthat the nucleic acid is able to enter a plant cell(s), e.g., via an invivo or ex vivo protocol. By “in vivo,” it is meant in the nucleic acidis administered to a living body of a plant e.g. infiltration. By “exvivo” it is meant that cells or explants are modified outside of theplant, and then such cells or organs are regenerated to a plant. Anumber of vectors suitable for stable transformation of plant cells orfor the establishment of transgenic plants have been described,including those described in Weissbach and Weissbach, (1989) Methods forPlant Molecular Biology Academic Press, and Gelvin et al., (1990) PlantMolecular Biology Manual, Kluwer Academic Publishers. Specific examplesinclude those derived from a Ti plasmid of Agrobacterium tumefaciens, aswell as those disclosed by Herrera-Estrella et al. (1983) Nature 303:209, Bevan (1984) Nucl Acid Res. 12: 8711-8721, Klee (1985) Bio/Technolo3: 637-642. Alternatively, non-Ti vectors can be used to transfer theDNA into plants and cells by using free DNA delivery techniques. Byusing these methods transgenic plants such as wheat, rice (Christou(1991) Bio/Technology 9:957-9 and 4462) and corn (Gordon-Kamm (1990)Plant Cell 2: 603-618) can be produced. An immature embryo can also be agood target tissue for monocots for direct DNA delivery techniques byusing the particle gun (Weeks et al. (1993) Plant Physiol 102:1077-1084; Vasil (1993) Bio/Technolo 10: 667-674; Wan and Lemeaux (1994)Plant Physiol 104: 37-48 and for Agrobacterium-mediated DNA transfer(Ishida et al. (1996) Nature Biotech 14: 745-750). Exemplary methods forintroduction of DNA into chloroplasts are biolistic bombardment,polyethylene glycol transformation of protoplasts, and microinjection(Danieli et al Nat. Biotechnol 16:345-348, 1998; Staub et al Nat.Biotechnol 18: 333-338, 2000; O'Neill et al Plant J. 3:729-738, 1993;Knoblauch et al Nat. Biotechnol 17: 906-909; U.S. Pat. Nos. 5,451,513,5,545,817, 5,545,818, and 5,576,198; in Intl. Application No. WO95/16783; and in Boynton et al., Methods in Enzymology 217: 510-536(1993), Svab et al., Proc. Natl. Acad. Sci. USA 90: 913-917 (1993), andMcBride et al., Proc. Nati. Acad. Sci. USA 91: 7301-7305 (1994)). Anyvector suitable for the methods of biolistic bombardment, polyethyleneglycol transformation of protoplasts and microinjection will be suitableas a targeting vector for chloroplast transformation. Any doublestranded DNA vector may be used as a transformation vector, especiallywhen the method of introduction does not utilize Agrobacterium.

Plants which can be genetically modified include grains, forage crops,fruits, vegetables, oil seed crops, palms, forestry, and vines. Specificexamples of plants which can be modified follow: maize, banana, peanut,field peas, sunflower, tomato, canola, tobacco, wheat, barley, oats,potato, soybeans, cotton, carnations, sorghum, lupin and rice.

Also provided by the subject invention are transformed plant cells,tissues, plants and products that contain the transformed plant cells. Afeature of the subject transformed cells, and tissues and products thatinclude the same is the presence of a subject nucleic acid integratedinto the genome, and production by plant cells of a site-directedmodifying polypeptide, e.g., a naturally occurring Cas9; a modified,i.e., mutated or variant, Cas9; a chimeric Cas9; etc. Recombinant plantcells of the present invention are useful as populations of recombinantcells, or as a tissue, seed, whole plant, stem, fruit, leaf, root,flower, stem, tuber, grain, animal feed, a field of plants, and thelike.

A nucleic acid comprising a nucleotide sequence encoding a site-directedmodifying polypeptide (e.g., a naturally occurring Cas9; a modified,i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) can be under thecontrol of (i.e., operably linked to) an unknown promoter (e.g., whenthe nucleic acid randomly integrates into a host cell genome) or can beunder the control of (i.e., operably linked to) a known promoter.Suitable known promoters can be any known promoter and includeconstitutively active promoters, inducible promoters, spatiallyrestricted and/or temporally restricted promoters, etc.

In some cases, the site-directed modifying polypeptide comprises anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, or 100%, amino acid sequence identity to amino acids 7-166 or731-1003 of the Cas9/Csn1 amino acid sequence depicted in FIG. 3A andFIG. 3B, or to the corresponding portions in any of the amino acidsequences set forth as SEQ ID NOs:1-256 and 795-1346.

Also provided by the subject invention is reproductive material of asubject transgenic plant, where reproductive material includes seeds,progeny plants and clonal material.

DEFINITIONS—PART II

The term “naturally-occurring” or “unmodified” as used herein as appliedto a nucleic acid, a polypeptide, a cell, or an organism, refers to anucleic acid, polypeptide, cell, or organism that is found in nature.For example, a polypeptide or polynucleotide sequence that is present inan organism (including viruses) that can be isolated from a source innature and which has not been intentionally modified by a human in thelaboratory is naturally occurring.

“Heterologous,” as used herein, means a nucleotide or polypeptidesequence that is not found in the native nucleic acid or protein,respectively. For example, in a fusion variant Cas9 site-directedpolypeptide, a variant Cas9 site-directed polypeptide may be fused to aheterologous polypeptide (i.e. a polypeptide other than Cas9). Theheterologous polypeptide may exhibit an activity (e.g., enzymaticactivity) that will also be exhibited by the fusion variant Cas9site-directed polypeptide. A heterologous nucleic acid sequence may belinked to a variant Cas9 site-directed polypeptide (e.g., by geneticengineering) to generate a nucleotide sequence encoding a fusion variantCas9 site-directed polypeptide.

The term “chimeric polypeptide” refers to a polypeptide which is notnaturally occurring, e.g., is made by the artificial combination of twootherwise separated segments of amino sequence through humanintervention. Thus, a chimeric polypeptide is also the result of humanintervention. Thus, a polypeptide that comprises a chimeric amino acidsequence is a chimeric polypeptide.

By “site-directed polypeptide” or “RNA-binding site-directedpolypeptide” or “RNA-binding site-directed polypeptide” it is meant apolypeptide that binds RNA and is targeted to a specific DNA sequence. Asite-directed polypeptide as described herein is targeted to a specificDNA sequence by the RNA molecule to which it is bound. The RNA moleculecomprises a sequence that is complementary to a target sequence withinthe target DNA, thus targeting the bound polypeptide to a specificlocation within the target DNA (the target sequence).

In some embodiments, a subject nucleic acid (e.g., a DNA-targeting RNA,a nucleic acid comprising a nucleotide sequence encoding a DNA-targetingRNA; a nucleic acid encoding a site-directed polypeptide; etc.)comprises a modification or sequence that provides for an additionaldesirable feature (e.g., modified or regulated stability; subcellulartargeting; tracking, e.g., a fluorescent label; a binding site for aprotein or protein complex; etc.). Non-limiting examples include: a 5′cap (e.g., a 7-methylguanylate cap (m⁷G)); a 3′ polyadenylated tail(i.e., a 3′ poly(A) tail); a riboswitch sequence (e.g., to allow forregulated stability and/or regulated accessibility by proteins and/orprotein complexes); a modification or sequence that targets the RNA to asubcellular location (e.g., nucleus, mitochondria, chloroplasts, and thelike); a modification or sequence that provides for tracking (e.g.,direct conjugation to a fluorescent molecule, conjugation to a moietythat facilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.); a modification or sequence that provides abinding site for proteins (e.g., proteins that act on DNA, includingtranscriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like); and combinations thereof.

In some embodiments, a DNA-targeting RNA comprises an additional segmentat either the 5′ or 3′ end that provides for any of the featuresdescribed above. For example, a suitable third segment can comprise a 5′cap (e.g., a 7-methylguanylate cap (m⁷G)); a 3′ polyadenylated tail(i.e., a 3′ poly(A) tail); a riboswitch sequence (e.g., to allow forregulated stability and/or regulated accessibility by proteins andprotein complexes); a sequence that targets the RNA to a subcellularlocation (e.g., nucleus, mitochondria, chloroplasts, and the like); amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.); a modification or sequence that provides abinding site for proteins (e.g., proteins that act on DNA, includingtranscriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like); and combinations thereof.

A subject DNA-targeting RNA and a subject site-directed polypeptide forma complex (i.e., bind via non-covalent interactions). The DNA-targetingRNA provides target specificity to the complex by comprising anucleotide sequence that is complementary to a sequence of a target DNA.The site-directed polypeptide of the complex provides the site-specificactivity. In other words, the site-directed polypeptide is guided to atarget DNA sequence (e.g. a target sequence in a chromosomal nucleicacid; a target sequence in an extrachromosomal nucleic acid, e.g. anepisomal nucleic acid, a minicircle, etc.; a target sequence in amitochondrial nucleic acid; a target sequence in a chloroplast nucleicacid; a target sequence in a plasmid; etc.) by virtue of its associationwith the protein-binding segment of the DNA-targeting RNA.

In some embodiments, a subject DNA-targeting RNA comprises two separateRNA molecules (RNA polynucleotides) and is referred to herein as a“double-molecule DNA-targeting RNA” or a “two-molecule DNA-targetingRNA.” In other embodiments, a subject DNA-targeting RNA is a single RNAmolecule (single RNA polynucleotide) and is referred to herein as a“single-molecule DNA-targeting RNA.”. If not otherwise specified, theterm “DNA-targeting RNA” is inclusive, referring to both single-moleculeDNA-targeting RNAs and double-molecule DNA-targeting RNAs.

A subject two-molecule DNA-targeting RNA comprises two separate RNAmolecules (a “targeter-RNA” and an “activator-RNA”). Each of the two RNAmolecules of a subject two-molecule DNA-targeting RNA comprises astretch of nucleotides that are complementary to one another such thatthe complementary nucleotides of the two RNA molecules hybridize to formthe double stranded RNA duplex of the protein-binding segment.

A subject single-molecule DNA-targeting RNA comprises two stretches ofnucleotides (a targeter-RNA and an activator-RNA) that are complementaryto one another, are covalently linked by intervening nucleotides(“linkers” or “linker nucleotides”), and hybridize to form the doublestranded RNA duplex (dsRNA duplex) of the protein-binding segment, thusresulting in a stem-loop structure. The targeter-RNA and theactivator-RNA can be covalently linked via the 3′ end of thetargeter-RNA and the 5′ end of the activator-RNA. Alternatively,targeter-RNA and the activator-RNA can be covalently linked via the 5′end of the targeter-RNA and the 3′ end of the activator-RNA.

An exemplary two-molecule DNA-targeting RNA comprises a crRNA-like(“CRISPR RNA” or “targeter-RNA” or “crRNA” or “crRNA repeat”) moleculeand a corresponding tracrRNA-like (“trans-acting CRISPR RNA” or“activator-RNA” or “tracrRNA”) molecule. A crRNA-like molecule(targeter-RNA) comprises both the DNA-targeting segment (singlestranded) of the DNA-targeting RNA and a stretch (“duplex-formingsegment”) of nucleotides that forms one half of the dsRNA duplex of theprotein-binding segment of the DNA-targeting RNA. A correspondingtracrRNA-like molecule (activator-RNA) comprises a stretch ofnucleotides (duplex-forming segment) that forms the other half of thedsRNA duplex of the protein-binding segment of the DNA-targeting RNA. Inother words, a stretch of nucleotides of a crRNA-like molecule arecomplementary to and hybridize with a stretch of nucleotides of atracrRNA-like molecule to form the dsRNA duplex of the protein-bindingdomain of the DNA-targeting RNA. As such, each crRNA-like molecule canbe said to have a corresponding tracrRNA-like molecule. The crRNA-likemolecule additionally provides the single stranded DNA-targetingsegment. Thus, a crRNA-like and a tracrRNA-like molecule (as acorresponding pair) hybridize to form a DNA-targeting RNA. The exactsequence of a given crRNA or tracrRNA molecule is characteristic of thespecies in which the RNA molecules are found.

The term “activator-RNA” is used herein to mean a tracrRNA-like moleculeof a double-molecule DNA-targeting RNA. The term “targeter-RNA” is usedherein to mean a crRNA-like molecule of a double-molecule DNA-targetingRNA. The term “duplex-forming segment” is used herein to mean thestretch of nucleotides of an activator-RNA or a targeter-RNA thatcontributes to the formation of the dsRNA duplex by hybridizing to astretch of nucleotides of a corresponding activator-RNA or targeter-RNAmolecule. In other words, an activator-RNA comprises a duplex-formingsegment that is complementary to the duplex-forming segment of thecorresponding targeter-RNA. As such, an activator-RNA comprises aduplex-forming segment while a targeter-RNA comprises both aduplex-forming segment and the DNA-targeting segment of theDNA-targeting RNA. Therefore, a subject double-molecule DNA-targetingRNA can be comprised of any corresponding activator-RNA and targeter-RNApair.

A two-molecule DNA-targeting RNA can be designed to allow for controlled(i.e., conditional) binding of a targeter-RNA with an activator-RNA.Because a two-molecule DNA-targeting RNA is not functional unless boththe activator-RNA and the targeter-RNA are bound in a functional complexwith dCas9, a two-molecule DNA-targeting RNA can be inducible (e.g.,drug inducible) by rendering the binding between the activator-RNA andthe targeter-RNA to be inducible. As one non-limiting example, RNAaptamers can be used to regulate (i.e., control) the binding of theactivator-RNA with the targeter-RNA. Accordingly, the activator-RNAand/or the targeter-RNA can comprise an RNA aptamer sequence.

RNA aptamers are known in the art and are generally a synthetic versionof a riboswitch. The terms “RNA aptamer” and “riboswitch” are usedinterchangeably herein to encompass both synthetic and natural nucleicacid sequences that provide for inducible regulation of the structure(and therefore the availability of specific sequences) of the RNAmolecule of which they are part. RNA aptamers usually comprise asequence that folds into a particular structure (e.g., a hairpin), whichspecifically binds a particular drug (e.g., a small molecule). Bindingof the drug causes a structural change in the folding of the RNA, whichchanges a feature of the nucleic acid of which the aptamer is a part. Asnon-limiting examples: (i) an activator-RNA with an aptamer may not beable to bind to the cognate targeter-RNA unless the aptamer is bound bythe appropriate drug; (ii) a targeter-RNA with an aptamer may not beable to bind to the cognate activator-RNA unless the aptamer is bound bythe appropriate drug; and (iii) a targeter-RNA and an activator-RNA,each comprising a different aptamer that binds a different drug, may notbe able to bind to each other unless both drugs are present. Asillustrated by these examples, a two-molecule DNA-targeting RNA can bedesigned to be inducible.

Examples of aptamers and riboswitches can be found, for example, in:Nakamura et al., Genes Cells. 2012 May; 17(5):344-64; Vavalle et al.,Future Cardiol. 2012 May; 8(3):371-82; Citartan et al., BiosensBioelectron. 2012 Apr. 15; 34(1):1-11; and Liberman et al., WileyInterdiscip Rev RNA. 2012 May-June; 3(3):369-84; all of which are hereinincorporated by reference in their entirety.

Non-limiting examples of nucleotide sequences that can be included in atwo-molecule DNA-targeting RNA include targeter RNAs (e.g., SEQ IDNOs:566-567) that can pair with the duplex forming seqment of any one ofthe activator RNAs set forth in SEQ ID NOs:671-678.

An exemplary single-molecule DNA-targeting RNA comprises twocomplementary stretches of nucleotides that hybridize to form a dsRNAduplex. In some embodiments, one of the two complementary stretches ofnucleotides of the single-molecule DNA-targeting RNA (or the DNAencoding the stretch) is at least about 60% identical to one of theactivator-RNA (tracrRNA) sequences set forth in SEQ ID NOs:431-562 overa stretch of at least 8 contiguous nucleotides. For example, one of thetwo complementary stretches of nucleotides of the single-moleculeDNA-targeting RNA (or the DNA encoding the stretch) is at least about65% identical, at least about 70% identical, at least about 75%identical, at least about 80% identical, at least about 85% identical,at least about 90% identical, at least about 95% identical, at leastabout 98% identical, at least about 99% identical or 100% identical toone of the tracrRNA sequences set forth in SEQ ID NOs:431-562 over astretch of at least 8 contiguous nucleotides.

In some embodiments, one of the two complementary stretches ofnucleotides of the single-molecule DNA-targeting RNA (or the DNAencoding the stretch) is at least about 60% identical to one of thetargeter-RNA (crRNA) sequences set forth in SEQ ID NOs:563-679 over astretch of at least 8 contiguous nucleotides. For example, one of thetwo complementary stretches of nucleotides of the single-moleculeDNA-targeting RNA (or the DNA encoding the stretch) is at least about65% identical, at least about 70% identical, at least about 75%identical, at least about 80% identical, at least about 85% identical,at least about 90% identical, at least about 95% identical, at leastabout 98% identical, at least about 99% identical or 100% identical toone of the crRNA sequences set forth in SEQ ID NOs:563-679 over astretch of at least 8 contiguous nucleotides.

As above, a “host cell,” as used herein, denotes an in vivo or in vitroeukaryotic cell, a prokaryotic cell (e.g., bacterial or archaeal cell),or a cell from a multicellular organism (e.g., a cell line) cultured asa unicellular entity, which eukaryotic or prokaryotic cells can be, orhave been, used as recipients for a nucleic acid, and include theprogeny of the original cell which has been transformed by the nucleicacid. It is understood that the progeny of a single cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement as the original parent, due to natural, accidental, ordeliberate mutation. A “recombinant host cell” (also referred to as a“genetically modified host cell”) is a host cell into which has beenintroduced a heterologous nucleic acid, e.g., an expression vector. Forexample, a subject bacterial host cell is a genetically modifiedbacterial host cell by virtue of introduction into a suitable bacterialhost cell of an exogenous nucleic acid (e.g., a plasmid or recombinantexpression vector) and a subject eukaryotic host cell is a geneticallymodified eukaryotic host cell (e.g., a mammalian germ cell), by virtueof introduction into a suitable eukaryotic host cell of an exogenousnucleic acid.

Definitions provided in “Definitions—Part I” are also applicable to theinstant section; see “Definitions—Part I” for additional clarificationof terms.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anenzymatically inactive Cas9 polypeptide” includes a plurality of suchpolypeptides and reference to “the target nucleic acid” includesreference to one or more target nucleic acids and equivalents thereofknown to those skilled in the art, and so forth. It is further notedthat the claims may be drafted to exclude any optional element. As such,this statement is intended to serve as antecedent basis for use of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION—PART II

The present disclosure provides methods of modulating transcription of atarget nucleic acid in a host cell. The methods generally involvecontacting the target nucleic acid with an enzymatically inactive Cas9polypeptide and a single-guide RNA. The methods are useful in a varietyof applications, which are also provided.

A transcriptional modulation method of the present disclosure overcomessome of the drawbacks of methods involving RNAi. A transcriptionalmodulation method of the present disclosure finds use in a wide varietyof applications, including research applications, drug discovery (e.g.,high throughput screening), target validation, industrial applications(e.g., crop engineering; microbial engineering, etc.), diagnosticapplications, therapeutic applications, and imaging techniques.

Methods of Modulating Transcription

The present disclosure provides a method of selectively modulatingtranscription of a target DNA in a host cell. The method generallyinvolves: a) introducing into the host cell: i) a DNA-targeting RNA, ora nucleic acid comprising a nucleotide sequence encoding theDNA-targeting RNA; and ii) a variant Cas9 site-directed polypeptide(“variant Cas9 polypeptide”), or a nucleic acid comprising a nucleotidesequence encoding the variant Cas9 polypeptide, where the variant Cas9polypeptide exhibits reduced endodeoxyribonuclease activity.

The DNA-targeting RNA (also referred to herein as “crRNA”; or “guideRNA”; or “gRNA”) comprises: i) a first segment comprising a nucleotidesequence that is complementary to a target sequence in a target DNA; ii)a second segment that interacts with a site-directed polypeptide; andiii) a transcriptional terminator. The first segment, comprising anucleotide sequence that is complementary to a target sequence in atarget DNA, is referred to herein as a “targeting segment”. The secondsegment, which interacts with a site-directed polypeptide, is alsoreferred to herein as a “protein-binding sequence” or “dCas9-bindinghairpin,” or “dCas9 handle.” By “segment” it is meant asegment/section/region of a molecule, e.g., a contiguous stretch ofnucleotides in an RNA. The definition of “segment,” unless otherwisespecifically defined in a particular context, is not limited to aspecific number of total base pairs, and may include regions of RNAmolecules that are of any total length and may or may not includeregions with complementarity to other molecules. A DNA-targeting RNAaccording to the present disclosure can be a single RNA molecule (singleRNA polynucleotide), which can be referred to herein as a“single-molecule DNA-targeting RNA,” a “single-guide RNA,” or an“sgRNA.” A DNA-targeting RNA according to the present disclosure cancomprise two RNA molecules. The term “DNA-targeting RNA” or “gRNA” isinclusive, referring both to two-molecule DNA-targeting RNAs and tosingle-molecule DNA-targeting RNAs (i.e., sgRNAs).

The variant Cas9 site-directed polypeptide comprises: i) an RNA-bindingportion that interacts with the DNA-targeting RNA; and ii) an activityportion that exhibits reduced endodeoxyribonuclease activity.

The DNA-targeting RNA and the variant Cas9 polypeptide form a complex inthe host cell; the complex selectively modulates transcription of atarget DNA in the host cell.

In some cases, a transcription modulation method of the presentdisclosure provides for selective modulation (e.g., reduction orincrease) of a target nucleic acid in a host cell. For example,“selective” reduction of transcription of a target nucleic acid reducestranscription of the target nucleic acid by at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, or greater than 90%, compared to the level of transcriptionof the target nucleic acid in the absence of a DNA-targeting RNA/variantCas9 polypeptide complex. Selective reduction of transcription of atarget nucleic acid reduces transcription of the target nucleic acid,but does not substantially reduce transcription of a non-target nucleicacid, e.g., transcription of a non-target nucleic acid is reduced, if atall, by less than 10% compared to the level of transcription of thenon-target nucleic acid in the absence of the DNA-targeting RNA/variantCas9 polypeptide complex.

Increased Transcription

“Selective” increased transcription of a target DNA can increasetranscription of the target DNA by at least about 1.1 fold (e.g., atleast about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold,at least about 1.5 fold, at least about 1.6 fold, at least about 1.7fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5fold, at least about 4 fold, at least about 4.5 fold, at least about 5fold, at least about 6 fold, at least about 7 fold, at least about 8fold, at least about 9 fold, at least about 10 fold, at least about 12fold, at least about 15 fold, or at least about 20-fold) compared to thelevel of transcription of the target DNA in the absence of aDNA-targeting RNA/variant Cas9 polypeptide complex. Selective increaseof transcription of a target DNA increases transcription of the targetDNA, but does not substantially increase transcription of a non-targetDNA, e.g., transcription of a non-target DNA is increased, if at all, byless than about 5-fold (e.g., less than about 4-fold, less than about3-fold, less than about 2-fold, less than about 1.8-fold, less thanabout 1.6-fold, less than about 1.4-fold, less than about 1.2-fold, orless than about 1.1-fold) compared to the level of transcription of thenon-targeted DNA in the absence of the DNA-targeting RNA/variant Cas9polypeptide complex.

As a non-limiting example, increased can be achieved by fusing dCas9 toa heterologous sequence. Suitable fusion partners include, but are notlimited to, a polypeptide that provides an activity that indirectlyincreases transcription by acting directly on the target DNA or on apolypeptide (e.g., a histone or other DNA-binding protein) associatedwith the target DNA. Suitable fusion partners include, but are notlimited to, a polypeptide that provides for methyltransferase activity,demethylase activity, acetyltransferase activity, deacetylase activity,kinase activity, phosphatase activity, ubiquitin ligase activity,deubiquitinating activity, adenylation activity, deadenylation activity,SUMOylating activity, deSUMOylating activity, ribosylation activity,deribosylation activity, myristoylation activity, or demyristoylationactivity.

Additional suitable fusion partners include, but are not limited to, apolypeptide that directly provides for increased transcription of thetarget nucleic acid (e.g., a transcription activator or a fragmentthereof, a protein or fragment thereof that recruits a transcriptionactivator, a small molecule/drug-responsive transcription regulator,etc.).

A non-limiting example of a subject method using a dCas9 fusion proteinto increase transcription in a prokaryote includes a modification of thebacterial one-hybrid (B1H) or two-hybrid (B2H) system. In the B1Hsystem, a DNA binding domain (BD) is fused to a bacterial transcriptionactivation domain (AD, e.g., the alpha subunit of the Escherichia coliRNA polymerase (RNAPa)). Thus, a subject dCas9 can be fused to aheterologous sequence comprising an AD. When the subject dCas9 fusionprotein arrives at the upstream region of a promoter (targeted there bythe DNA-targeting RNA) the AD (e.g., RNAPa) of the dCas9 fusion proteinrecruits the RNAP holoenzyme, leading to transcription activation. Inthe B2H system, the BD is not directly fused to the AD; instead, theirinteraction is mediated by a protein-protein interaction (e.g.,GAL11P-GAL4 interaction). To modify such a system for use in the subjectmethods, dCas9 can be fused to a first protein sequence that providesfor protein-protein interaction (e.g., the yeast GALI1P and/or GAL4protein) and RNAa can be fused to a second protein sequence thatcompletes the protein-protein interaction (e.g., GAL4 if GALI1P is fusedto dCas9, GALI1P if GAL4 is fused to dCas9, etc.). The binding affinitybetween GALI1P and GAL4 increases the efficiency of binding andtranscription firing rate.

A non-limiting example of a subject method using a dCas9 fusion proteinto increase transcription in a eukaryotes includes fusion of dCas9 to anactivation domain (AD) (e.g., GAL4, herpesvirus activation protein VP16or VP64, human nuclear factor NF-κB p65 subunit, etc.). To render thesystem inducible, expression of the dCas9 fusion protein can becontrolled by an inducible promoter (e.g., Tet-ON, Tet-OFF, etc.). TheDNA-targeting RNA can be design to target known transcription responseelements (e.g., promoters, enhancers, etc.), known upstream activatingsequences (UAS), sequences of unknown or known function that aresuspected of being able to control expression of the target DNA, etc.

Additional Fusion Partners

Non-limiting examples of fusion partners to accomplish increased ordecreased transcription are listed in FIG. 54A-54C and includetranscription activator and transcription repressor domains (e.g., theKruppel associated box (KRAB or SKD); the Mad mSIN3 interaction domain(SID); the ERF repressor domain (ERD), etc). In some such cases, thedCas9 fusion protein is targeted by the DNA-targeting RNA to a specificlocation (i.e., sequence) in the target DNA and exerts locus-specificregulation such as blocking RNA polymerase binding to a promoter (whichselectively inhibits transcription activator function), and/or modifyingthe local chromatin status (e.g., when a fusion sequence is used thatmodifies the target DNA or modifies a polypeptide associated with thetarget DNA). In some cases, the changes are transient (e.g.,transcription repression or activation). In some cases, the changes areinheritable (e.g., when epigenetic modifications are made to the targetDNA or to proteins associated with the target DNA, e.g., nucleosomalhistones).

In some embodiments, the heterologous sequence can be fused to theC-terminus of the dCas9 polypeptide. In some embodiments, theheterologous sequence can be fused to the N-terminus of the dCas9polypeptide. In some embodiments, the heterologous sequence can be fusedto an internal portion (i.e., a portion other than the N- or C-terminus)of the dCas9 polypeptide.

The biological effects of a method using a subject dCas9 fusion proteincan be detected by any convenient method (e.g., gene expression assays;chromatin-based assays, e.g., Chromatin immunoPrecipitation (ChiP),Chromatin in vivo Assay (CiA), etc.; and the like).

In some cases, a subject method involves use of two or more differentDNA-targeting RNAs. For example, two different DNA-targeting RNAs can beused in a single host cell, where the two different DNA-targeting RNAstarget two different target sequences in the same target nucleic acid.

Thus, for example, a subject transcriptional modulation method canfurther comprise introducing into the host cell a second DNA-targetingRNA, or a nucleic acid comprising a nucleotide sequence encoding thesecond DNA-targeting RNA, where the second DNA-targeting RNA comprises:i) a first segment comprising a nucleotide sequence that iscomplementary to a second target sequence in the target DNA; ii) asecond segment that interacts with the site-directed polypeptide; andiii) a transcriptional terminator. In some cases, use of two differentDNA-targeting RNAs targeting two different targeting sequences in thesame target nucleic acid provides for increased modulation (e.g.,reduction or increase) in transcription of the target nucleic acid.

As another example, two different DNA-targeting RNAs can be used in asingle host cell, where the two different DNA-targeting RNAs target twodifferent target nucleic acids. Thus, for example, a subjecttranscriptional modulation method can further comprise introducing intothe host cell a second DNA-targeting RNA, or a nucleic acid comprising anucleotide sequence encoding the second DNA-targeting RNA, where thesecond DNA-targeting RNA comprises: i) a first segment comprising anucleotide sequence that is complementary to a target sequence in atleast a second target DNA; ii) a second segment that interacts with thesite-directed polypeptide; and iii) a transcriptional terminator.

In some embodiments, a subject nucleic acid (e.g., a DNA-targeting RNA,e.g., a single-molecule DNA-targeting RNA, an activator-RNA, atargeter-RNA, etc.; a donor polynucleotide; a nucleic acid encoding asite-directed modifying polypeptide; etc.) comprises a modification orsequence that provides for an additional desirable feature (e.g.,modified or regulated stability; subcellular targeting; tracking, e.g.,a fluorescent label; a binding site for a protein or protein complex;etc.). Non-limiting examples include: a 5′ cap (e.g., a7-methylguanylate cap (m⁷G)); a 3′ polyadenylated tail (i.e., a 3′poly(A) tail); a riboswitch sequence or an aptamer sequence (e.g., toallow for regulated stability and/or regulated accessibility by proteinsand/or protein complexes); a terminator sequence; a sequence that formsa dsRNA duplex (i.e., a hairpin)); a modification or sequence thattargets the RNA to a subcellular location (e.g., nucleus, mitochondria,chloroplasts, and the like); a modification or sequence that providesfor tracking (e.g., direct conjugation to a fluorescent molecule,conjugation to a moiety that facilitates fluorescent detection, asequence that allows for fluorescent detection, etc.); a modification orsequence that provides a binding site for proteins (e.g., proteins thatact on DNA, including transcriptional activators, transcriptionalrepressors, DNA methyltransferases, DNA demethylases, histoneacetyltransferases, histone deacetylases, and the like); andcombinations thereof.

DNA-Targeting Segment

The DNA-targeting segment (or “DNA-targeting sequence”) of aDNA-targeting RNA (“crRNA”) comprises a nucleotide sequence that iscomplementary to a specific sequence within a target DNA (thecomplementary strand of the target DNA).

In other words, the DNA-targeting segment of a subject DNA-targeting RNAinteracts with a target DNA in a sequence-specific manner viahybridization (i.e., base pairing). As such, the nucleotide sequence ofthe DNA-targeting segment may vary and determines the location withinthe target DNA that the DNA-targeting RNA and the target DNA willinteract. The DNA-targeting segment of a subject DNA-targeting RNA canbe modified (e.g., by genetic engineering) to hybridize to any desiredsequence within a target DNA.

The DNA-targeting segment can have a length of from about 12 nucleotidesto about 100 nucleotides. For example, the DNA-targeting segment canhave a length of from about 12 nucleotides (nt) to about 80 nt, fromabout 12 nt to about 50 nt, from about 12 nt to about 40 nt, from about12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 ntto about 20 nt, or from about 12 nt to about 19 nt. For example, theDNA-targeting segment can have a length of from about 19 nt to about 20nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt,from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, fromabout 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about19 nt to about 60 nt, from about 19 nt to about 70 nt, from about 19 ntto about 80 nt, from about 19 nt to about 90 nt, from about 19 nt toabout 100 nt, from about 20 nt to about 25 nt, from about 20 nt to about30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt,from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, fromabout 20 nt to about 60 nt, from about 20 nt to about 70 nt, from about20 nt to about 80 nt, from about 20 nt to about 90 nt, or from about 20nt to about 100 nt.

The nucleotide sequence (the DNA-targeting sequence) of theDNA-targeting segment that is complementary to a nucleotide sequence(target sequence) of the target DNA can have a length at least about 12nt. For example, the DNA-targeting sequence of the DNA-targeting segmentthat is complementary to a target sequence of the target DNA can have alength at least about 12 nt, at least about 15 nt, at least about 18 nt,at least about 19 nt, at least about 20 nt, at least about 25 nt, atleast about 30 nt, at least about 35 nt or at least about 40 nt. Forexample, the DNA-targeting sequence of the DNA-targeting segment that iscomplementary to a target sequence of the target DNA can have a lengthof from about 12 nucleotides (nt) to about 80 nt, from about 12 nt toabout 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt,from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, fromabout 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 ntto about 35 nt, from about 19 nt to about 40 nt, from about 19 nt toabout 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt,from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, fromabout 20 nt to about 45 nt, from about 20 nt to about 50 nt, or fromabout 20 nt to about 60 nt. The nucleotide sequence (the DNA-targetingsequence) of the DNA-targeting segment that is complementary to anucleotide sequence (target sequence) of the target DNA can have alength at least about 12 nt.

In some cases, the DNA-targeting sequence of the DNA-targeting segmentthat is complementary to a target sequence of the target DNA is 20nucleotides in length. In some cases, the DNA-targeting sequence of theDNA-targeting segment that is complementary to a target sequence of thetarget DNA is 19 nucleotides in length.

The percent complementarity between the DNA-targeting sequence of theDNA-targeting segment and the target sequence of the target DNA can beat least 60% (e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, at least 99%, or 100%). In some cases, the percent complementaritybetween the DNA-targeting sequence of the DNA-targeting segment and thetarget sequence of the target DNA is 100% over the seven contiguous5′-most nucleotides of the target sequence of the complementary strandof the target DNA. In some cases, the percent complementarity betweenthe DNA-targeting sequence of the DNA-targeting segment and the targetsequence of the target DNA is at least 60% over about 20 contiguousnucleotides. In some cases, the percent complementarity between theDNA-targeting sequence of the DNA-targeting segment and the targetsequence of the target DNA is 100% over the fourteen contiguous 5′-mostnucleotides of the target sequence of the complementary strand of thetarget DNA and as low as 0% over the remainder. In such a case, theDNA-targeting sequence can be considered to be 14 nucleotides in length.In some cases, the percent complementarity between the DNA-targetingsequence of the DNA-targeting segment and the target sequence of thetarget DNA is 100% over the seven contiguous 5′-most nucleotides of thetarget sequence of the complementary strand of the target DNA and as lowas 0% over the remainder. In such a case, the DNA-targeting sequence canbe considered to be 7 nucleotides in length.

Protein-Binding Segment

The protein-binding segment (i.e., “protein-binding sequence”) of aDNA-targeting RNA interacts with a variant site-directed polypeptide.When the variant Cas9 site-directed polypeptide, together with theDNA-targeting RNA, binds to a target DNA, transcription of the targetDNA is reduced.

The protein-binding segment of a DNA-targeting RNA comprises twocomplementary stretches of nucleotides that hybridize to one another toform a double stranded RNA duplex (dsRNA duplex).

The protein-binding segment of a DNA-targeting RNA of the presentdisclosure comprises two stretches of nucleotides (a targeter-RNA and anactivator-RNA) that are complementary to one another, are covalentlylinked by intervening nucleotides (e.g., in the case of asingle-molecule DNA-targeting RNA)(“linkers” or “linker nucleotides”),and hybridize to form the double stranded RNA duplex (dsRNA duplex, or“dCas9-binding hairpin”) of the protein-binding segment, thus resultingin a stem-loop structure. This stem-loop structure is shownschematically in FIG. 39A. The targeter-RNA and the activator-RNA can becovalently linked via the 3′ end of the targeter-RNA and the 5′ end ofthe activator-RNA. Alternatively, targeter-RNA and the activator-RNA canbe covalently linked via the 5′ end of the targeter-RNA and the 3′ endof the activator-RNA.

The protein-binding segment can have a length of from about 10nucleotides to about 100 nucleotides, e.g., from about 10 nucleotides(nt) to about 20 nt, from about 20 nt to about 30 nt, from about 30 ntto about 40 nt, from about 40 nt to about 50 nt, from about 50 nt toabout 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100nt. For example, the protein-binding segment can have a length of fromabout 15 nucleotides (nt) to about 80 nt, from about 15 nt to about 50nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt orfrom about 15 nt to about 25 nt.

The dsRNA duplex of the protein-binding segment can have a length fromabout 6 base pairs (bp) to about 50 bp. For example, the dsRNA duplex ofthe protein-binding segment can have a length from about 6 bp to about40 bp, from about 6 bp to about 30 bp, from about 6 bp to about 25 bp,from about 6 bp to about 20 bp, from about 6 bp to about 15 bp, fromabout 8 bp to about 40 bp, from about 8 bp to about 30 bp, from about 8bp to about 25 bp, from about 8 bp to about 20 bp or from about 8 bp toabout 15 bp. For example, the dsRNA duplex of the protein-bindingsegment can have a length from about from about 8 bp to about 10 bp,from about 10 bp to about 15 bp, from about 15 bp to about 18 bp, fromabout 18 bp to about 20 bp, from about 20 bp to about 25 bp, from about25 bp to about 30 bp, from about 30 bp to about 35 bp, from about 35 bpto about 40 bp, or from about 40 bp to about 50 bp. In some embodiments,the dsRNA duplex of the protein-binding segment has a length of 36 basepairs. The percent complementarity between the nucleotide sequences thathybridize to form the dsRNA duplex of the protein-binding segment can beat least about 60%. For example, the percent complementarity between thenucleotide sequences that hybridize to form the dsRNA duplex of theprotein-binding segment can be at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or at least about99%. In some cases, the percent complementarity between the nucleotidesequences that hybridize to form the dsRNA duplex of the protein-bindingsegment is 100%.

The linker can have a length of from about 3 nucleotides to about 100nucleotides. For example, the linker can have a length of from about 3nucleotides (nt) to about 90 nt, from about 3 nucleotides (nt) to about80 nt, from about 3 nucleotides (nt) to about 70 nt, from about 3nucleotides (nt) to about 60 nt, from about 3 nucleotides (nt) to about50 nt, from about 3 nucleotides (nt) to about 40 nt, from about 3nucleotides (nt) to about 30 nt, from about 3 nucleotides (nt) to about20 nt or from about 3 nucleotides (nt) to about 10 nt. For example, thelinker can have a length of from about 3 nt to about 5 nt, from about 5nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt toabout 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt,from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, fromabout 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about80 nt to about 90 nt, or from about 90 nt to about 100 nt. In someembodiments, the linker of a DNA-targeting RNA is 4 nt.

Non-limiting examples of nucleotide sequences that can be included in asuitable protein-binding segment (i.e., dCas9 handle) are set forth inSEQ ID NOs:563-682 (For examples, see FIG. 8 and FIG. 9).

In some cases, a suitable protein-binding segment comprises a nucleotidesequence that differs by 1, 2, 3, 4, or 5 nucleotides from any one ofthe above-listed sequences.

Stability Control Sequence (e.g., Transcriptional Terminator Segment)

A stability control sequence influences the stability of an RNA (e.g., aDNA-targeting RNA, a targeter-RNA, an activator-RNA, etc.). One exampleof a suitable stability control sequence is a transcriptional terminatorsegment (i.e., a transcription termination sequence). A transcriptionalterminator segment of a subject DNA-targeting RNA can have a totallength of from about 10 nucleotides to about 100 nucleotides, e.g., fromabout 10 nucleotides (nt) to about 20 nt, from about 20 nt to about 30nt, from about 30 nt to about 40 nt, from about 40 nt to about 50 nt,from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, fromabout 70 nt to about 80 nt, from about 80 nt to about 90 nt, or fromabout 90 nt to about 100 nt. For example, the transcriptional terminatorsegment can have a length of from about 15 nucleotides (nt) to about 80nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt.

In some cases, the transcription termination sequence is one that isfunctional in a eukaryotic cell. In some cases, the transcriptiontermination sequence is one that is functional in a prokaryotic cell.

Non-limiting examples of nucleotide sequences that can be included in astability control sequence (e.g., transcriptional termination segment,or in any segment of the DNA-targeting RNA to provide for increasedstability) include sequences set forth in SEQ ID NO:683-696 and, forexample, 5′-UAAUCCCACAGCCGCCAGUUCCGCUGGCGGCAUUUU-5′ (SEQ ID NO: 1349) (aRho-independent trp termination site).

Additional Sequences

In some embodiments, a DNA-targeting RNA comprises at least oneadditional segment at either the 5′ or 3′ end. For example, a suitableadditional segment can comprise a 5′ cap (e.g., a 7-methylguanylate cap(m⁷G)); a 3′ polyadenylated tail (i.e., a 3′ poly(A) tail); a riboswitchsequence (e.g., to allow for regulated stability and/or regulatedaccessibility by proteins and protein complexes); a sequence that formsa dsRNA duplex (i.e., a hairpin)); a sequence that targets the RNA to asubcellular location (e.g., nucleus, mitochondria, chloroplasts, and thelike); a modification or sequence that provides for tracking (e.g.,direct conjugation to a fluorescent molecule, conjugation to a moietythat facilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.); a modification or sequence that provides abinding site for proteins (e.g., proteins that act on DNA, includingtranscriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like) a modification or sequence thatprovides for increased, decreased, and/or controllable stability; andcombinations thereof.

Multiple Simultaneous DNA-Targeting RNAs

In some embodiments, multiple DNA-targeting RNAs are used simultaneouslyin the same cell to simultaneously modulate transcription at differentlocations on the same target DNA or on different target DNAs. In someembodiments, two or more DNA-targeting RNAs target the same gene ortranscript or locus. In some embodiments, two or more DNA-targeting RNAstarget different unrelated loci. In some embodiments, two or moreDNA-targeting RNAs target different, but related loci.

Because the DNA-targeting RNAs are small and robust they can besimultaneously present on the same expression vector and can even beunder the same transcriptional control if so desired. In someembodiments, two or more (e.g., 3 or more, 4 or more, 5 or more, 10 ormore, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 ormore, 45 or more, or 50 or more) DNA-targeting RNAs are simultaneouslyexpressed in a target cell (from the same or different vectors). Theexpressed DNA-targeting RNAs can be differently recognized by dCas9proteins from different bacteria, such as S. pyogenes, S. thermophilus,L. innocua, and N. meningitidis.

To express multiple DNA-targeting RNAs, an artificial RNA processingsystem mediated by the Csy4 endoribonuclease can be used. MultipleDNA-targeting RNAs can be concatenated into a tandem array on aprecursor transcript (e.g., expressed from a U6 promoter), and separatedby Csy4-specific RNA sequence. Co-expressed Csy4 protein cleaves theprecursor transcript into multiple DNA-targeting RNAs. Advantages forusing an RNA processing system include: first, there is no need to usemultiple promoters; second, since all DNA-targeting RNAs are processedfrom a precursor transcript, their concentrations are normalized forsimilar dCas9-binding.

Csy4 is a small endoribonuclease (RNase) protein derived from bacteriaPseudomonas aeruginosa. Csy4 specifically recognizes a minimal 17-bp RNAhairpin, and exhibits rapid (<1 min) and highly efficient (>99.9%) RNAcleavage. Unlike most RNases, the cleaved RNA fragment remains stableand functionally active. The Csy4-based RNA cleavage can be repurposedinto an artificial RNA processing system. In this system, the 17-bp RNAhairpins are inserted between multiple RNA fragments that aretranscribed as a precursor transcript from a single promoter.Co-expression of Csy4 is effective in generating individual RNAfragments.

Site-Directed Polypeptide

As noted above, a subject DNA-targeting RNA and a variant Cas9site-directed polypeptide form a complex. The DNA-targeting RNA providestarget specificity to the complex by comprising a nucleotide sequencethat is complementary to a sequence of a target DNA.

The variant Cas9 site-directed polypeptide has reducedendodeoxyribonuclease activity. For example, a variant Cas9site-directed polypeptide suitable for use in a transcription modulationmethod of the present disclosure exhibits less than about 20%, less thanabout 15%, less than about 10%, less than about 5%, less than about 1%,or less than about 0.1%, of the endodeoxyribonuclease activity of awild-type Cas9 polypeptide, e.g., a wild-type Cas9 polypeptidecomprising an amino acid sequence as depicted in FIG. 3A and FIG. 3B(SEQ ID NO:8). In some embodiments, the variant Cas9 site-directedpolypeptide has substantially no detectable endodeoxyribonucleaseactivity. In some embodiments when a site-directed polypeptide hasreduced catalytic activity (e.g., when a Cas9 protein has a D10, G12,G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or a A987mutation, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A,H983A, A984A, and/or D986A), the polypeptide can still bind to targetDNA in a site-specific manner (because it is still guided to a targetDNA sequence by a DNA-targeting RNA) as long as it retains the abilityto interact with the DNA-targeting RNA.

In some cases, a suitable variant Cas9 site-directed polypeptidecomprises an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 99% or 100% amino acid sequence identity to amino acids7-166 or 731-1003 of the Cas9/Csn1 amino acid sequence depicted in FIG.3A and FIG. 3B (SEQ ID NO:8), or to the corresponding portions in anyone of the amino acid sequences SEQ ID NOs:1-256 and 795-1346.

In some cases, the variant Cas9 site-directed polypeptide can cleave thecomplementary strand of the target DNA but has reduced ability to cleavethe non-complementary strand of the target DNA. For example, the variantCas9 site-directed polypeptide can have a mutation (amino acidsubstitution) that reduces the function of the RuvC domain (e.g.,“domain 1” of FIG. 3B). As a non-limiting example, in some cases, thevariant Cas9 site-directed polypeptide is a D10A (aspartate to alanine)mutation of the amino acid sequence depicted in FIG. 3A and FIG. 3B (orthe corresponding mutation of any of the amino acid sequences set forthin SEQ ID NOs:1-256 and 795-1346).

In some cases, the variant Cas9 site-directed polypeptide can cleave thenon-complementary strand of the target DNA but has reduced ability tocleave the complementary strand of the target DNA. For example, thevariant Cas9 site-directed polypeptide can have a mutation (amino acidsubstitution) that reduces the function of the HNH domain (RuvC/HNH/RuvCdomain motifs, “domain 2” of FIG. 3B). As a non-limiting example, insome cases, the variant Cas9 site-directed polypeptide is a H840A(histidine to alanine at amino acid position 840 of SEQ ID NO:8) or thecorresponding mutation of any of the amino acid sequences set forth inSEQ ID NOs:1-256 and 795-1346).

In some cases, the variant Cas9 site-directed polypeptide has a reducedability to cleave both the complementary and the non-complementarystrands of the target DNA. As a non-limiting example, in some cases, thevariant Cas9 site-directed polypeptide harbors both D10A and H840Amutations of the amino acid sequence depicted in FIG. 3A and FIG. 3B (orthe corresponding mutations of any of the amino acid sequences set forthin SEQ ID NOs:1-256 and 795-1346).

Other residues can be mutated to achieve the same effect (i.e.inactivate one or the other nuclease portions). As non-limitingexamples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983,A984, D986, and/or A987 (or the corresponding mutations of any of theproteins set forth as SEQ ID NOs:1-256 and 795-1346) can be altered(i.e., substituted) (see FIG. 3A-3B, FIG. 5, FIG. 11A, and Table 1 formore information regarding the conservation of Cas9 amino acidresidues). Also, mutations other than alanine substitutions aresuitable.

In some cases, the variant Cas9 site-directed polypeptide is a fusionpolypeptide (a “variant Cas9 fusion polypeptide”), i.e., a fusionpolypeptide comprising: i) a variant Cas9 site-directed polypeptide; andb) a covalently linked heterologous polypeptide (also referred to as a“fusion partner”).

The heterologous polypeptide may exhibit an activity (e.g., enzymaticactivity) that will also be exhibited by the variant Cas9 fusionpolypeptide (e.g., methyltransferase activity, acetyltransferaseactivity, kinase activity, ubiquitinating activity, etc.). Aheterologous nucleic acid sequence may be linked to another nucleic acidsequence (e.g., by genetic engineering) to generate a chimericnucleotide sequence encoding a chimeric polypeptide. In someembodiments, a variant Cas9 fusion polypeptide is generated by fusing avariant Cas9 polypeptide with a heterologous sequence that provides forsubcellular localization (i.e., the heterologous sequence is asubcellular localization sequence, e.g., a nuclear localization signal(NLS) for targeting to the nucleus; a mitochondrial localization signalfor targeting to the mitochondria; a chloroplast localization signal fortargeting to a chloroplast; an ER retention signal; and the like). Insome embodiments, the heterologous sequence can provide a tag (i.e., theheterologous sequence is a detectable label) for ease of tracking and/orpurification (e.g., a fluorescent protein, e.g., green fluorescentprotein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like; ahistidine tag, e.g., a 6×His tag; a hemagglutinin (HA) tag; a FLAG tag;a Myc tag; and the like). In some embodiments, the heterologous sequencecan provide for increased or decreased stability (i.e., the heterologoussequence is a stability control peptide, e.g., a degron, which in somecases is controllable (e.g., a temperature sensitive or drugcontrollable degron sequence, see below). In some embodiments, theheterologous sequence can provide for increased or decreasedtranscription from the target DNA (i.e., the heterologous sequence is atranscription modulation sequence, e.g., a transcriptionfactor/activator or a fragment thereof, a protein or fragment thereofthat recruits a transcription factor/activator, a transcriptionrepressor or a fragment thereof, a protein or fragment thereof thatrecruits a transcription repressor, a small molecule/drug-responsivetranscription regulator, etc.). In some embodiments, the heterologoussequence can provide a binding domain (i.e., the heterologous sequenceis a protein binding sequence, e.g., to provide the ability of achimeric dCas9 polypeptide to bind to another protein of interest, e.g.,a DNA or histone modifying protein, a transcription factor ortranscription repressor, a recruiting protein, etc.).

Suitable fusion partners that provide for increased or decreasedstability include, but are not limited to degron sequences. Degrons arereadily understood by one of ordinary skill in the art to be amino acidsequences that control the stability of the protein of which they arepart. For example, the stability of a protein comprising a degronsequence is controlled at least in part by the degron sequence. In somecases, a suitable degron is constitutive such that the degron exerts itsinfluence on protein stability independent of experimental control(i.e., the degron is not drug inducible, temperature inducible, etc.) Insome cases, the degron provides the variant Cas9 polypeptide withcontrollable stability such that the variant Cas9 polypeptide can beturned “on” (i.e., stable) or “off” (i.e., unstable, degraded) dependingon the desired conditions. For example, if the degron is a temperaturesensitive degron, the variant Cas9 polypeptide may be functional (i.e.,“on”, stable) below a threshold temperature (e.g., 42° C., 41° C., 40°C., 39° C., 38° C., 37° C., 36° C., 35° C., 34° C., 33° C., 32° C., 31°C., 30° C., etc.) but non-functional (i.e., “off”, degraded) above thethreshold temperature. As another example, if the degron is a druginducible degron, the presence or absence of drug can switch the proteinfrom an “off” (i.e., unstable) state to an “on” (i.e., stable) state orvice versa. An exemplary drug inducible degron is derived from theFKBP12 protein. The stability of the degron is controlled by thepresence or absence of a small molecule that binds to the degron.

Examples of suitable degrons include, but are not limited to thosedegrons controlled by Shield-1, DHFR, auxins, and/or temperature.Non-limiting examples of suitable degrons are known in the art (e.g.,Dohmen et al., Science, 1994. 263(5151): p. 1273-1276: Heat-inducibledegron: a method for constructing temperature-sensitive mutants;Schoeber et al., Am J Physiol Renal Physiol. 2009 January;296(1):F204-11: Conditional fast expression and function of multimericTRPV5 channels using Shield-1; Chu et al., Bioorg Med Chem Lett. 2008Nov. 15; 18(22):5941-4: Recent progress with FKBP-derived destabilizingdomains; Kanemaki, Pflugers Arch. 2012 Dec. 28: Frontiers of proteinexpression control with conditional degrons; Yang et al., Mol Cell. 2012Nov. 30; 48(4):487-8: Titivated for destruction: the methyl degron;Barbour et al., Biosci Rep. 2013 Jan. 18; 33(1): Characterization of thebipartite degron that regulates ubiquitin-independent degradation ofthymidylate synthase; and Greussing et al., J Vis Exp. 2012 Nov. 10;(69): Monitoring of ubiquitin-proteasome activity in living cells usinga Degron (dgn)-destabilized green fluorescent protein (GFP)-basedreporter protein; all of which are hereby incorporated in their entiretyby reference).

Exemplary degron sequences have been well-characterized and tested inboth cells and animals. Thus, fusing dCas9 to a degron sequence producesa “tunable” and “inducible” dCas9 polypeptide. Any of the fusionpartners described herein can be used in any desirable combination. Asone non-limiting example to illustrate this point, a dCas9 fusionprotein can comprise a YFP sequence for detection, a degron sequence forstability, and transcription activator sequence to increasetranscription of the target DNA. Furthermore, the number of fusionpartners that can be used in a dCas9 fusion protein is unlimited. Insome cases, a dCas9 fusion protein comprises one or more (e.g. two ormore, three or more, four or more, or five or more) heterologoussequences.

Suitable fusion partners include, but are not limited to, a polypeptidethat provides for methyltransferase activity, demethylase activity,acetyltransferase activity, deacetylase activity, kinase activity,phosphatase activity, ubiquitin ligase activity, deubiquitinatingactivity, adenylation activity, deadenylation activity, SUMOylatingactivity, deSUMOylating activity, ribosylation activity, deribosylationactivity, myristoylation activity, or demyristoylation activity, any ofwhich can be directed at modifying the DNA directly (e.g., methylationof DNA) or at modifying a DNA-associated polypeptide (e.g., a histone orDNA binding protein). Further suitable fusion partners include, but arenot limited to boundary elements (e.g., CTCF), proteins and fragmentsthereof that provide periphery recruitment (e.g., Lamin A, Lamin B,etc.), and protein docking elements (e.g., FKBP/FRB, Pill/Abyl, etc.).

Examples of various additional suitable fusion partners (or fragmentsthereof) for a subject variant Cas9 site-directed polypeptide include,but are not limited to those listed in FIG. 54A-54C.

In some embodiments, a subject site-directed modifying polypeptide canbe codon-optimized. This type of optimization is known in the art andentails the mutation of foreign-derived DNA to mimic the codonpreferences of the intended host organism or cell while encoding thesame protein. Thus, the codons are changed, but the encoded proteinremains unchanged. For example, if the intended target cell was a humancell, a human codon-optimized dCas9 (or dCas9 variant) would be asuitable site-directed modifying polypeptide. As another non-limitingexample, if the intended host cell were a mouse cell, than a mousecodon-optimized Cas9 (or variant, e.g., enzymatically inactive variant)would be a suitable Cas9 site-directed polypeptide. While codonoptimization is not required, it is acceptable and may be preferable incertain cases.

Host Cells

A method of the present disclosure to modulate transcription may beemployed to induce transcriptional modulation in mitotic or post-mitoticcells in vivo and/or ex vivo and/or in vitro. Because the DNA-targetingRNA provides specificity by hybridizing to target DNA, a mitotic and/orpost-mitotic cell can be any of a variety of host cell, where suitablehost cells include, but are not limited to, a bacterial cell; anarchaeal cell; a single-celled eukaryotic organism; a plant cell; analgal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii,Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.agardh, and the like; a fungal cell; an animal cell; a cell from aninvertebrate animal (e.g., an insect, a cnidarian, an echinoderm, anematode, etc.); a eukaryotic parasite (e.g., a malarial parasite, e.g.,Plasmodium falciparum; a helminth; etc.); a cell from a vertebrateanimal (e.g., fish, amphibian, reptile, bird, mammal); a mammalian cell,e.g., a rodent cell, a human cell, a non-human primate cell, etc.Suitable host cells include naturally-occurring cells; geneticallymodified cells (e.g., cells genetically modified in a laboratory, e.g.,by the “hand of man”); and cells manipulated in vitro in any way. Insome cases, a host cell is isolated.

Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonicstem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell; asomatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, amuscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitroor in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell,2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.). Cells may befrom established cell lines or they may be primary cells, where “primarycells”, “primary cell lines”, and “primary cultures” are usedinterchangeably herein to refer to cells and cells cultures that havebeen derived from a subject and allowed to grow in vitro for a limitednumber of passages, i.e. splittings, of the culture. For example,primary cultures include cultures that may have been passaged 0 times, 1time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enoughtimes go through the crisis stage. Primary cell lines can be aremaintained for fewer than 10 passages in vitro. Target cells are in manyembodiments unicellular organisms, or are grown in culture.

If the cells are primary cells, such cells may be harvest from anindividual by any convenient method. For example, leukocytes may beconveniently harvested by apheresis, leukocytapheresis, density gradientseparation, etc., while cells from tissues such as skin, muscle, bonemarrow, spleen, liver, pancreas, lung, intestine, stomach, etc. are mostconveniently harvested by biopsy. An appropriate solution may be usedfor dispersion or suspension of the harvested cells. Such solution willgenerally be a balanced salt solution, e.g. normal saline,phosphate-buffered saline (PBS), Hank's balanced salt solution, etc.,conveniently supplemented with fetal calf serum or other naturallyoccurring factors, in conjunction with an acceptable buffer at lowconcentration, e.g., from 5-25 mM. Convenient buffers include HEPES,phosphate buffers, lactate buffers, etc. The cells may be usedimmediately, or they may be stored, frozen, for long periods of time,being thawed and capable of being reused. In such cases, the cells willusually be frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40%buffered medium, or some other such solution as is commonly used in theart to preserve cells at such freezing temperatures, and thawed in amanner as commonly known in the art for thawing frozen cultured cells.

Introducing Nucleic Acid into a Host Cell

A DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequenceencoding same, can be introduced into a host cell by any of a variety ofwell-known methods. Similarly, where a subject method involvesintroducing into a host cell a nucleic acid comprising a nucleotidesequence encoding a variant Cas9 site-directed polypeptide, such anucleic acid can be introduced into a host cell by any of a variety ofwell-known methods.

Methods of introducing a nucleic acid into a host cell are known in theart, and any known method can be used to introduce a nucleic acid (e.g.,an expression construct) into a stem cell or progenitor cell. Suitablemethods include, include e.g., viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, calcium phosphate precipitation, polyethyleneimine(PEI)-mediated transfection, DEAE-dextran mediated transfection,liposome-mediated transfection, particle gun technology, calciumphosphate precipitation, direct micro injection, nanoparticle-mediatednucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev.2012 Sep. 13. pii: S0169-409X(12)00283-9. doi:10.1016/j.addr.2012.09.023), and the like.

Nucleic Acids

The present disclosure provides an isolated nucleic acid comprising anucleotide sequence encoding a subject DNA-targeting RNA. In some cases,a subject nucleic acid also comprises a nucleotide sequence encoding avariant Cas9 site-directed polypeptide.

In some embodiments, a subject method involves introducing into a hostcell (or a population of host cells) one or more nucleic acidscomprising nucleotide sequences encoding a DNA-targeting RNA and/or avariant Cas9 site-directed polypeptide. In some embodiments a cellcomprising a target DNA is in vitro. In some embodiments a cellcomprising a target DNA is in vivo. Suitable nucleic acids comprisingnucleotide sequences encoding a DNA-targeting RNA and/or a site-directedpolypeptide include expression vectors, where an expression vectorcomprising a nucleotide sequence encoding a DNA-targeting RNA and/or asite-directed polypeptide is a “recombinant expression vector.”

In some embodiments, the recombinant expression vector is a viralconstruct, e.g., a recombinant adeno-associated virus construct (see,e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, arecombinant lentiviral construct, a recombinant retroviral construct,etc.

Suitable expression vectors include, but are not limited to, viralvectors (e.g. viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549,1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like.

Numerous suitable expression vectors are known to those of skill in theart, and many are commercially available. The following vectors areprovided by way of example; for eukaryotic host cells: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, anyother vector may be used so long as it is compatible with the host cell.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

In some embodiments, a nucleotide sequence encoding a DNA-targeting RNAand/or a variant Cas9 site-directed polypeptide is operably linked to acontrol element, e.g., a transcriptional control element, such as apromoter. The transcriptional control element may be functional ineither a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell(e.g., bacterial or archaeal cell). In some embodiments, a nucleotidesequence encoding a DNA-targeting RNA and/or a variant Cas9site-directed polypeptide is operably linked to multiple controlelements that allow expression of the nucleotide sequence encoding aDNA-targeting RNA and/or a variant Cas9 site-directed polypeptide inboth prokaryotic and eukaryotic cells.

A promoter can be a constitutively active promoter (i.e., a promoterthat is constitutively in an active/“ON” state), it may be an induciblepromoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”,is controlled by an external stimulus, e.g., the presence of aparticular temperature, compound, or protein), it may be a spatiallyrestricted promoter (i.e., transcriptional control element, enhancer,etc.)(e.g., tissue specific promoter, cell type specific promoter,etc.), and it may be a temporally restricted promoter (i.e., thepromoter is in the “ON” state or “OFF” state during specific stages ofembryonic development or during specific stages of a biological process,e.g., hair follicle cycle in mice).

Suitable promoters can be derived from viruses and can therefore bereferred to as viral promoters, or they can be derived from anyorganism, including prokaryotic or eukaryotic organisms. Suitablepromoters can be used to drive expression by any RNA polymerase (e.g.,pol I, pol II, pol III). Exemplary promoters include, but are notlimited to the SV40 early promoter, mouse mammary tumor virus longterminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP);a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE), a rous sarcomavirus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishiet al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), ahuman H1 promoter (H1), and the like.

Examples of inducible promoters include, but are not limited toT7 RNApolymerase promoter, T3 RNA polymerase promoter,Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter,lactose induced promoter, heat shock promoter, Tetracycline-regulatedpromoter (e.g., Tet-ON, Tet-OFF, etc.), Steroid-regulated promoter,Metal-regulated promoter, estrogen receptor-regulated promoter, etc.Inducible promoters can therefore be regulated by molecules including,but not limited to, doxycycline; RNA polymerase, e.g., T7 RNApolymerase; an estrogen receptor; an estrogen receptor fusion; etc.

In some embodiments, the promoter is a spatially restricted promoter(i.e., cell type specific promoter, tissue specific promoter, etc.) suchthat in a multi-cellular organism, the promoter is active (i.e., “ON”)in a subset of specific cells. Spatially restricted promoters may alsobe referred to as enhancers, transcriptional control elements, controlsequences, etc. Any convenient spatially restricted promoter may be usedand the choice of suitable promoter (e.g., a brain specific promoter, apromoter that drives expression in a subset of neurons, a promoter thatdrives expression in the germline, a promoter that drives expression inthe lungs, a promoter that drives expression in muscles, a promoter thatdrives expression in islet cells of the pancreas, etc.) will depend onthe organism. For example, various spatially restricted promoters areknown for plants, flies, worms, mammals, mice, etc. Thus, a spatiallyrestricted promoter can be used to regulate the expression of a nucleicacid encoding a subject site-directed polypeptide in a wide variety ofdifferent tissues and cell types, depending on the organism. Somespatially restricted promoters are also temporally restricted such thatthe promoter is in the “ON” state or “OFF” state during specific stagesof embryonic development or during specific stages of a biologicalprocess (e.g., hair follicle cycle in mice).

For illustration purposes, examples of spatially restricted promotersinclude, but are not limited to, neuron-specific promoters,adipocyte-specific promoters, cardiomyocyte-specific promoters, smoothmuscle-specific promoters, photoreceptor-specific promoters, etc.Neuron-specific spatially restricted promoters include, but are notlimited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBLHSENO2, X51956); an aromatic amino acid decarboxylase (AADC) promoter; aneurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsinpromoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see,e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat.Med. 16(10):1161-1166); a serotonin receptor promoter (see, e.g.,GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh etal. (2009) Gene Ther 16:437; Sasaoka et al. (1992) Mol. Brain Res.16:274; Boundy et al. (1998) J. Neurosci. 18:9989; and Kaneda et al.(1991) Neuron 6:583-594); a GnRH promoter (see, e.g., Radovick et al.(1991) Proc. Natl. Acad. Sci. USA 88:3402-3406); an L7 promoter (see,e.g., Oberdick et al. (1990) Science 248:223-226); a DNMT promoter (see,e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); anenkephalin promoter (see, e.g., Comb et al. (1988) EMBO J.17:3793-3805); a myelin basic protein (MBP) promoter; aCa²⁺-calmodulin-dependent protein kinase II-alpha (CamKIIα) promoter(see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250;and Casanova et al. (2001) Genesis 31:37); a CMVenhancer/platelet-derived growth factor-O promoter (see, e.g., Liu etal. (2004) Gene Therapy 11:52-60); and the like.

Adipocyte-specific spatially restricted promoters include, but are notlimited to aP2 gene promoter/enhancer, e.g., a region from −5.4 kb to+21 bp of a human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol.138:1604; Ross et al. (1990) Proc. Natl. Acad. Sci. USA 87:9590; andPavjani et al. (2005) Nat. Med. 11:797); a glucose transporter-4 (GLUT4)promoter (see, e.g., Knight et al. (2003) Proc. Natl. Acad. Sci. USA100:14725); a fatty acid translocase (FAT/CD36) promoter (see, e.g.,Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato et al. (2002)J. Biol. Chem. 277:15703); a stearoyl-CoA desaturase-1 (SCD1) promoter(Tabor et al. (1999) J. Biol. Chem. 274:20603); a leptin promoter (see,e.g., Mason et al. (1998) Endocrinol. 139:1013; and Chen et al. (1999)Biochem. Biophys. Res. Comm. 262:187); an adiponectin promoter (see,e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm. 331:484; andChakrabarti (2010) Endocrinol. 151:2408); an adipsin promoter (see,e.g., Platt et al. (1989) Proc. Natl. Acad. Sci. USA 86:7490); aresistin promoter (see, e.g., Seo et al. (2003) Molec. Endocrinol.17:1522); and the like.

Cardiomyocyte-specific spatially restricted promoters include, but arenot limited to control sequences derived from the following genes:myosin light chain-2, α-myosin heavy chain, AE3, cardiac troponin C,cardiac actin, and the like. Franz et al. (1997) Cardiovasc. Res.35:560-566; Robbins et al. (1995) Ann. N.Y. Acad. Sci. 752:492-505; Linnet al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Mol. Cell.Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; andSartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.

Smooth muscle-specific spatially restricted promoters include, but arenot limited to an SM22a promoter (see, e.g., Akyurek et al. (2000) Mol.Med. 6:983; and U.S. Pat. No. 7,169,874); a smoothelin promoter (see,e.g., WO 2001/018048); an α-smooth muscle actin promoter; and the like.For example, a 0.4 kb region of the SM22α promoter, within which lie twoCArG elements, has been shown to mediate vascular smooth musclecell-specific expression (see, e.g., Kim, et al. (1997) Mol. Cell. Biol.17, 2266-2278; Li, et al., (1996) J. Cell Biol. 132, 849-859; andMoessler, et al. (1996) Development 122, 2415-2425).

Photoreceptor-specific spatially restricted promoters include, but arenot limited to, a rhodopsin promoter; a rhodopsin kinase promoter (Younget al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterasegene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); a retinitispigmentosa gene promoter (Nicoud et al. (2007) supra); aninterphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoudet al. (2007) supra); an IRBP gene promoter (Yokoyama et al. (1992) ExpEye Res. 55:225); and the like.

Libraries

The present disclosure provides a library of DNA-targeting RNAs. Thepresent disclosure provides a library of nucleic acids comprisingnucleotides encoding DNA-targeting RNAs. A subject library of nucleicacids comprising nucleotides encoding DNA-targeting RNAs can comprises alibrary of recombinant expression vectors comprising nucleotidesencoding the DNA-targeting RNAs.

A subject library can comprise from about 10 individual members to about10¹² individual members; e.g., a subject library can comprise from about10 individual members to about 10² individual members, from about 10²individual members to about 10³ individual members, from about 10³individual members to about 10⁵ individual members, from about 10⁵individual members to about 10⁷ individual members, from about 10⁷individual members to about 10⁹ individual members, or from about 10⁹individual members to about 10¹² individual members.

An “individual member” of a subject library differs from other membersof the library in the nucleotide sequence of the DNA targeting segmentof the DNA-targeting RNA. Thus, e.g., each individual member of asubject library can comprise the same or substantially the samenucleotide sequence of the protein-binding segment as all other membersof the library; and can comprise the same or substantially the samenucleotide sequence of the transcriptional termination segment as allother members of the library; but differs from other members of thelibrary in the nucleotide sequence of the DNA targeting segment of theDNA-targeting RNA. In this way, the library can comprise members thatbind to different target nucleic acids.

Utility

A method for modulating transcription according to the presentdisclosure finds use in a variety of applications, which are alsoprovided. Applications include research applications; diagnosticapplications; industrial applications; and treatment applications.

Research applications include, e.g., determining the effect of reducingor increasing transcription of a target nucleic acid on, e.g.,development, metabolism, expression of a downstream gene, and the like.

High through-put genomic analysis can be carried out using a subjecttranscription modulation method, in which only the DNA-targeting segmentof the DNA-targeting RNA needs to be varied, while the protein-bindingsegment and the transcription termination segment can (in some cases) beheld constant. A library (e.g., a subject library) comprising aplurality of nucleic acids used in the genomic analysis would include: apromoter operably linked to a DNA-targeting RNA-encoding nucleotidesequence, where each nucleic acid would include a differentDNA-targeting segment, a common protein-binding segment, and a commontranscription termination segment. A chip could contain over 5×10⁴unique DNA-targeting RNAs. Applications would include large-scalephenotyping, gene-to-function mapping, and meta-genomic analysis.

The subject methods disclosed herein find use in the field of metabolicengineering. Because transcription levels can be efficiently andpredictably controlled by designing an appropriate DNA-targeting RNA, asdisclosed herein, the activity of metabolic pathways (e.g., biosyntheticpathways) can be precisely controlled and tuned by controlling the levelof specific enzymes (e.g., via increased or decreased transcription)within a metabolic pathway of interest. Metabolic pathways of interestinclude those used for chemical (fine chemicals, fuel, antibiotics,toxins, agonists, antagonists, etc.) and/or drug production.

Biosynthetic pathways of interest include but are not limited to (1) themevalonate pathway (e.g., HMG-CoA reductase pathway) (convertsacetyl-CoA to dimethylallyl pyrophosphate (DMAPP) and isopentenylpyrophosphate (IPP), which are used for the biosynthesis of a widevariety of biomolecules including terpenoids/isoprenoids), (2) thenon-mevalonate pathway (i.e., the “2-C-methyl-D-erythritol4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway” or “MEP/DOXPpathway” or “DXP pathway”)(also produces DMAPP and IPP, instead byconverting pyruvate and glyceraldehyde 3-phosphate into DMAPP and IPPvia an alternative pathway to the mevalonate pathway), (3) thepolyketide synthesis pathway (produces a variety of polyketides via avariety of polyketide synthase enzymes. Polyketides include naturallyoccurring small molecules used for chemotherapy (e.g., tetracyclin, andmacrolides) and industrially important polyketides include rapamycin(immunosuppressant), erythromycin (antibiotic), lovastatin(anticholesterol drug), and epothilone B (anticancer drug)), (4) fattyacid synthesis pathways, (5) the DAHP (3-deoxy-D-arabino-heptulosonate7-phosphate) synthesis pathway, (6) pathways that produce potentialbiofuels (such as short-chain alcohols and alkane, fatty acid methylesters and fatty alcohols, isoprenoids, etc.), etc.

Networks and Cascades

The methods disclosed herein can be used to design integrated networks(i.e., a cascade or cascades) of control. For example, a subjectDNA-targeting RNA/variant Cas9 site-directed polypeptide may be used tocontrol (i.e., modulate, e.g., increase, decrease) the expression ofanother DNA-targeting RNA or another subject variant Cas9 site-directedpolypeptide. For example, a first DNA-targeting RNA may be designed totarget the modulation of transcription of a second chimeric dCas9polypeptide with a function that is different than the first variantCas9 site-directed polypeptide (e.g., methyltransferase activity,demethylase activity, acetyltansferase activity, deacetylase activity,etc.). In addition, because different dCas9 proteins (e.g., derived fromdifferent species) may require a different Cas9 handle (i.e., proteinbinding segment), the second chimeric dCas9 polypeptide can be derivedfrom a different species than the first dCas9 polypeptide above. Thus,in some cases, the second chimeric dCas9 polypeptide can be selectedsuch that it may not interact with the first DNA-targeting RNA. In othercases, the second chimeric dCas9 polypeptide can be selected such thatit does interact with the first DNA-targeting RNA. In some such cases,the activities of the two (or more) dCas9 proteins may compete (e.g., ifthe polypeptides have opposing activities) or may synergize (e.g., ifthe polypeptides have similar or synergistic activities). Likewise, asnoted above, any of the complexes (i.e., DNA-targeting RNA/dCas9polypeptide) in the network can be designed to control otherDNA-targeting RNAs or dCas9 polypeptides. Because a subjectDNA-targeting RNA and subject variant Cas9 site-directed polypeptide canbe targeted to any desired DNA sequence, the methods described hereincan be used to control and regulate the expression of any desiredtarget. The integrated networks (i.e., cascades of interactions) thatcan be designed range from very simple to very complex, and are withoutlimit.

In a network wherein two or more components (e.g., DNA-targeting RNAs,activator-RNAs, targeter-RNAs, or dCas9 polypeptides) are each underregulatory control of another DNA-targeting RNA/dCas9 polypeptidecomplex, the level of expression of one component of the network mayaffect the level of expression (e.g., may increase or decrease theexpression) of another component of the network. Through this mechanism,the expression of one component may affect the expression of a differentcomponent in the same network, and the network may include a mix ofcomponents that increase the expression of other components, as well ascomponents that decrease the expression of other components. As would bereadily understood by one of skill in the art, the above exampleswhereby the level of expression of one component may affect the level ofexpression of one or more different component(s) are for illustrativepurposes, and are not limiting. An additional layer of complexity may beoptionally introduced into a network when one or more components aremodified (as described above) to be manipulable (i.e., underexperimental control, e.g., temperature control; drug control, i.e.,drug inducible control; light control; etc.).

As one non-limiting example, a first DNA-targeting RNA can bind to thepromoter of a second DNA-targeting RNA, which controls the expression ofa target therapeutic/metabolic gene. In such a case, conditionalexpression of the first DNA-targeting RNA indirectly activates thetherapeutic/metabolic gene. RNA cascades of this type are useful, forexample, for easily converting a repressor into an activator, and can beused to control the logics or dynamics of expression of a target gene.

A subject transcription modulation method can also be used for drugdiscovery and target validation.

Kits

The present disclosure provides a kit for carrying out a subject method.A subject kit comprises: a) a DNA-targeting RNA of the presentdisclosure, or a nucleic acid comprising a nucleotide sequence encodingthe DNA-targeting RNA, wherein the DNA-targeting RNA comprises: i)) afirst segment comprising a nucleotide sequence that is complementary toa target sequence in the target DNA; ii)) a second segment thatinteracts with a site-directed polypeptide; and iii) a transcriptionalterminator; and b) a buffer. In some cases, the nucleic acid comprisinga nucleotide sequence encoding the DNA-targeting RNA further comprises anucleotide sequence encoding a variant Cas9 site-directed polypeptidethat exhibits reduced endodeoxyribonuclease activity relative towild-type Cas9.

In some cases, a subject kit further comprises a variant Cas9site-directed polypeptide that exhibits reduced endodeoxyribonucleaseactivity relative to wild-type Cas9.

In some cases, a subject kit further comprises a nucleic acid comprisinga nucleotide sequence encoding a variant Cas9 site-directed polypeptidethat exhibits reduced endodeoxyribonuclease activity relative towild-type Cas9.

A subject can further include one or more additional reagents, wheresuch additional reagents can be selected from: a buffer; a wash buffer;a control reagent; a control expression vector or RNA polynucleotide; areagent for in vitro production of the variant Cas9 site-directedpolypeptide from DNA; and the like. In some cases, the variant Cas9site-directed polypeptide included in a subject kit is a fusion variantCas9 site-directed polypeptide, as described above.

Components of a subject kit can be in separate containers; or can becombined in a single container.

In addition to above-mentioned components, a subject kit can furtherinclude instructions for using the components of the kit to practice thesubject methods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Use of Cas9 to Generate Modifications in Target DNA

Materials and Methods

Bacterial Strains and Culture Conditions

Streptococcus pyogenes, cultured in THY medium (Todd Hewitt Broth (THB,Bacto, Becton Dickinson) supplemented with 0.2% yeast extract (Oxoid))or on TSA (trypticase soy agar, BBL, Becton Dickinson) supplemented with3% sheep blood, was incubated at 37° C. in an atmosphere supplementedwith 5% CO₂ without shaking. Escherichia coli, cultured in Luria-Bertani(LB) medium and agar, was incubated at 37° C. with shaking. Whenrequired, suitable antibiotics were added to the medium at the followingfinal concentrations: ampicillin, 100 μg/ml for E. coli;chloramphenicol, 33 μg/ml for Escherichia coli; kanamycin, 25 μg/ml forE. coli and 300 μg/ml for S. pyogenes. Bacterial cell growth wasmonitored periodically by measuring the optical density of culturealiquots at 620 nm using a microplate reader (SLT Spectra Reader).

Transformation of Bacterial Cells

Plasmid DNA transformation into E. coli cells was performed according toa standard heat shock protocol. Transformation of S. pyogenes wasperformed as previously described with some modifications. Thetransformation assay performed to monitor in vivo CRISPR/Cas activity onplasmid maintenance was essentially carried out as described previously.Briefly, electrocompetent cells of S. pyogenes were equalized to thesame cell density and electroporated with 500 ng of plasmid DNA. Everytransformation was plated two to three times and the experiment wasperformed three times independently with different batches of competentcells for statistical analysis. Transformation efficiencies werecalculated as CFU (colony forming units) per jg of DNA. Controltransformations were performed with sterile water and backbone vectorpEC85.

DNA Manipulations

DNA manipulations including DNA preparation, amplification, digestion,ligation, purification, agarose gel electrophoresis were performedaccording to standard techniques with minor modifications. Protospacerplasmids for the in vitro cleavage and S. pyogenes transformation assayswere constructed as described previously (4). Additional pUC19-basedprotospacer plasmids for in vitro cleavage assays were generated byligating annealed oligonucleotides between digested EcoRI and BamHIsites in pUC19. The GFP gene-containing plasmid has been describedpreviously (41). Kits (Qiagen) were used for DNA purification andplasmid preparation. Plasmid mutagenesis was performed using QuikChange®II XL kit (Stratagene) or QuikChange site-directed mutagenesis kit(Agilent). VBC-Biotech Services, Sigma-Aldrich and Integrated DNATechnologies supplied the synthetic oligonucleotides and RNAs.

Oligonucleotides for In Vitro Transcription Templates

Templates for in vitro transcribed CRISPR Type II-A tracrRNA and crRNAsof S. pyogenes (for tracrRNA—PCR on chr. DNA SF370; for crRNA—annealingof two oligonucleotides)

T7-tracrRNA (75 nt)

OLEC1521 (F 5′ tracrRNA): SEQ ID NO:340

OLEC1522 (R 3′ tracrRNA): SEQ ID NO:341

T7-crRNA (template)

OLEC2176 (F crRNA-sp1): SEQ ID NO:342

OLEC2178 (R crRNA-sp1): SEQ ID NO:343

OLEC2177 (F crRNA-sp2): SEQ ID NO:344

OLEC2179 (R crRNA-sp2): SEQ ID NO:345

Templates for in vitro transcribed N. meningitidis tracrRNA andengineered crRNA-sp2 (for tracrRNA—PCR on chr. DNA Z2491; forcrRNA—annealing of two oligonucleotides)

T7-tracrRNA

OLEC2205 (F predicted 5′): SEQ ID NO:346

OLEC2206 (R predicted 3′): SEQ ID NO:347

T7-crRNA (template)

OLEC2209 (F sp2(speM)+N.m. repeat): SEQ ID NO:348

OLEC2214 (R sp2(speM)+N.m. repeat): SEQ ID NO:349

Templates for in vitro transcribed L. innocua tracrRNA and engineeredcrRNA-sp2 (for tracrRNA—PCR on chr. DNA Clip11262; for crRNA—annealingof two oligonucleotides)

T7-tracrRNA

OLEC2203 (F predicted 5′): SEQ ID NO:350

OLEC2204 (R predicted 3′): SEQ ID NO:351

T7-crRNA (template)

OLEC2207 (F sp2(speM)+L.in. repeat): SEQ ID NO:352

OLEC2212 (R sp2(speM)+L.in. repeat): SEQ ID NO:353

Oligonucleotides for Constructing Plasmids with Protospacer for In Vitroand in Vivo Studies

Plasmids for speM (spacer 2 (CRISPR Type II-A, SF370; protospacerprophage 08232.3 from MGAS8232) analysis in vitro and in S. pyogenes(template: chr. DNA MGAS8232 or plasmids containing speM fragments)

pEC287

OLEC1555 (F speM): SEQ ID NO:354

OLEC1556 (R speM): SEQ ID NO:355

pEC488

OLEC2145 (F speM): SEQ ID NO:356

OLEC2146 (R speM): SEQ ID NO:357

pEC370

OLEC1593 (F pEC488 protospacer 2 A22G): SEQ ID NO:358

OLEC1594 (R pEC488 protospacer 2 A22G): SEQ ID NO:359

pEC371

OLEC1595 (F pEC488 protospacer 2 TIOC): SEQ ID NO:360

OLEC1596 (R pEC488 protospacer 2 TIOC): SEQ ID NO:361

pEC372

OLEC2185 (F pEC488 protospacer 2 T7A): SEQ ID NO:362

OLEC2186 (R pEC488 protospacer 2 T7A): SEQ ID NO:363

pEC373

OLEC2187 (F pEC488 protospacer 2 A6T): SEQ ID NO:364

OLEC2188 (R pEC488 protospacer 2 A6T): SEQ ID NO:365

pEC374

OLEC2235 (F pEC488 protospacer 2 A5T): SEQ ID NO:366

OLEC2236 (R pEC488 protospacer 2 A5T): SEQ ID NO:367

pEC375

OLEC2233 (F pEC488 protospacer 2 A4T): SEQ ID NO:368

OLEC2234 (R pEC488 protospacer 2 A4T): SEQ ID NO:369

pEC376

OLEC2189 (F pEC488 protospacer 2 A3T): SEQ ID NO:370

OLEC2190 (R pEC488 protospacer 2 A3T): SEQ ID NO:371

pEC377

OLEC2191 (F pEC488 protospacer 2 PAM G1C): SEQ ID NO:372

OLEC2192 (R pEC488 protospacer 2 PAM G1C): SEQ ID NO:373

pEC378

OLEC2237 (F pEC488 protospacer 2 PAM GG1, 2CC): SEQ ID NO:374

OLEC2238 (R pEC488 protospacer 2 PAM GG1, 2CC): SEQ ID NO:375

Plasmids for SPy_0700 (spacer 1 (CRISPR Type II-A, SF370; protospacerprophage ø0370.1 from SF370) analysis in vitro and in S. pyogenes(template: chr. DNA SF370 or plasmids containing SPy_0700 fragments)

pEC489

OLEC2106 (F Spy_0700): SEQ ID NO:376

OLEC2107 (R Spy_0700): SEQ ID NO:377

pEC573

OLEC2941 (F PAM TG1, 2GG): SEQ ID NO:378

OLEC2942 (R PAM TG1, 2GG): SEQ ID NO:379

Oligonucleotides for Verification of Plasmid Constructs and CuttingSites by Sequencing Analysis

ColE1 (pEC85)

oliRN228 (R sequencing): SEQ ID NO:380

speM (pEC287)

OLEC1557 (F sequencing): SEQ ID NO:381

OLEC1556 (R sequencing): SEQ ID NO:382

repDEG-pAMbeta1 (pEC85)

OLEC787 (F sequencing): SEQ ID NO:383

Oligonucleotides for In Vitro Cleavage Assays

crRNA

Spacer 1 crRNA (1-42): SEQ ID NO:384

Spacer 2 crRNA (1-42): SEQ ID NO:385

Spacer 4 crRNA (1-42): SEQ ID NO:386

Spacer 2 crRNA (1-36): SEQ ID NO:387

Spacer 2 crRNA (1-32): SEQ ID NO:388

Spacer 2 crRNA (11-42): SEQ ID NO:389

tracrRNA

(4-89): SEQ ID NO:390

(15-89): SEQ ID NO:391

(23-89): SEQ ID NO:392

(15-53): SEQ ID NO:393

(15-44): SEQ ID NO:394

(15-36): SEQ ID NO:395

(23-53): SEQ ID NO:396

(23-48): SEQ ID NO:397

(23-44): SEQ ID NO:398

(1-26): SEQ ID NO:399

chimeric RNAs

Spacer 1—chimera A: SEQ ID NO:400

Spacer 1—chimera B: SEQ ID NO:401

Spacer 2—chimera A: SEQ ID NO:402

Spacer 2—chimera B: SEQ ID NO:403

Spacer 4—chimera A: SEQ ID NO:404

Spacer 4—chimera B: SEQ ID NO:405

GFP1: SEQ ID NO:406

GFP2: SEQ ID NO:407

GFP3: SEQ ID NO:408

GFP4: SEQ ID NO:409

GFP5: SEQ ID NO:410

DNA Oligonucleotides as Substrates for Cleavage Assays (Protospacer inBold, PAM Underlined)

protospacer 1—complementary—WT: SEQ ID NO:411

protospacer 1—noncomplementary—WT: SEQ ID NO:412

protospacer 2—complementary—WT: SEQ ID NO:413

protospacer 2—noncomplementary—WT: SEQ ID NO:414

protospacer 4—complementary—WT: SEQ ID NO:415

protospacer 4—noncomplementary—WT: SEQ ID NO:416

protospacer 2—complementary—PAM1: SEQ ID NO:417

protospacer 2—noncomplementary—PAM1: SEQ ID NO:418

protospacer 2—complementary—PAM2: SEQ ID NO:419

protospacer 2—noncomplementary—PAM2: SEQ ID NO:420

protospacer 4—complementary—PAM1: SEQ ID NO:421

protospacer 4—noncomplementary—PAM1: SEQ ID NO:422

protospacer 4—complementary—PAM2: SEQ ID NO:423

protospacer 4—noncomplementary—PAM2: SEQ ID NO:424

In Vitro Transcription and Purification of RNA

RNA was in vitro transcribed using T7 Flash in vitro Transcription Kit(Epicentre, Illumina company) and PCR-generated DNA templates carrying aT7 promoter sequence. RNA was gel-purified and quality-checked prior touse. The primers used for the preparation of RNA templates from S.pyogenes SF370, Listeria innocua Clip 11262 and Neisseria meningitidis AZ2491 are described above.

Protein Purification

The sequence encoding Cas9 (residues 1-1368) was PCRamplified from thegenomic DNA of S. pyogenes SF370 and inserted into a custom pET-basedexpression vector using ligation-independent cloning (LIC). Theresulting fusion construct contained an N-terminal hexahistidine-maltosebinding protein (His6-MBP) tag, followed by a peptide sequencecontaining a tobacco etch virus (TEV) protease cleavage site. Theprotein was expressed in E. coli strain BL21 Rosetta 2 (DE3) (EMDBiosciences), grown in 2×TY medium at 18° C. for 16 h followinginduction with 0.2 mM IPTG. The protein was purified by a combination ofaffinity, ion exchange and size exclusion chromatographic steps.Briefly, cells were lysed in 20 mM Tris pH 8.0, 500 mM NaCl, 1 mM TCEP(supplemented with protease inhibitor cocktail (Roche)) in a homogenizer(Avestin). Clarified lysate was bound in batch to Ni-NTA agarose(Qiagen). The resin was washed extensively with 20 mM Tris pH 8.0, 500mM NaCl and the bound protein was eluted in 20 mM Tris pH 8.0, 250 mMNaCl, 10% glycerol. The His6-MBP affinity tag was removed by cleavagewith TEV protease, while the protein was dialyzed overnight against 20mM HEPES pH 7.5, 150 mM KCl, 1 mM TCEP, 10% glycerol. The cleaved Cas9protein was separated from the fusion tag by purification on a 5 ml SPSepharose HiTrap column (GE Life Sciences), eluting with a lineargradient of 100 mM-1 M KCl. The protein was further purified by sizeexclusion chromatography on a Superdex 200 16/60 column in 20 mM HEPESpH 7.5, 150 mM KCl and 1 mM TCEP. Eluted protein was concentrated to ˜8mg/ml, flash-frozen in liquid nitrogen and stored at −80° C. Cas9 D10A,H840A and D10A/H840A point mutants were generated using the QuikChangesite-directed mutagenesis kit (Agilent) and confirmed by DNA sequencing.The proteins were purified following the same procedure as for thewildtype Cas9 protein.

Cas9 orthologs from Streptococcus thermophilus (LMD-9,YP_820832.1), L.innocua (Clip11262, NP_472073.1), Campylobacter jejuni (subsp. jejuniNCTC 11168, YP_002344900.1) and N. meningitidis (Z2491, YP_002342100.1)were expressed in BL21 Rosetta (DE3) pLysS cells (Novagen) as His6-MBP(N. meningitidis and C. jejuni), His6-Thioredoxin (L. innocua) andHis6-GST (S. thermophilus) fusion proteins, and purified essentially asfor S. pyogenes Cas9 with the following modifications. Due to largeamounts of co-purifying nucleic acids, all four Cas9 proteins werepurified by an additional heparin sepharose step prior to gelfiltration, eluting the bound protein with a linear gradient of 100 mM-2M KCl. This successfully removed nucleic acid contamination from the C.jejuni, N. meningitidis and L. innocua proteins, but failed to removeco-purifying nucleic acids from the S. thermophilus Cas9 preparation.All proteins were concentrated to 1-8 mg/ml in 20 mM HEPES pH 7.5, 150mM KCl and 1 mM TCEP, flash-frozen in liquid N2 and stored at −80° C.

Plasmid DNA Cleavage Assay

Synthetic or in vitro-transcribed tracrRNA and crRNA were pre-annealedprior to the reaction by heating to 95° C. and slowly cooling down toroom temperature. Native or restriction digest-linearized plasmid DNA(300 ng (˜8 nM)) was incubated for 60 min at 37° C. with purified Cas9protein (50-500 nM) and tracrRNA:crRNA duplex (50-500 nM, 1:1) in a Cas9plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1mM EDTA) with or without 10 mM MgCl₂. The reactions were stopped with5×DNA loading buffer containing 250 mM EDTA, resolved by 0.8 or 1%agarose gel electrophoresis and visualized by ethidium bromide staining.For the Cas9 mutant cleavage assays, the reactions were stopped with5×SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA) prior toloading on the agarose gel.

Metal-Dependent Cleavage Assay

Protospacer 2 plasmid DNA (5 nM) was incubated for 1 h at 37° C. withCas9 (50 nM) pre-incubated with 50 nM tracrRNA:crRNA-sp2 in cleavagebuffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA)supplemented with 1, 5 or 10 mM MgCl₂, 1 or 10 mM of MnCl₂, CaCl₂,ZnCl₂, CoCl₂, NiSO₄ or CuSO₄. The reaction was stopped by adding 5×SDSloading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by 1%agarose gel electrophoresis and visualized by ethidium bromide staining.

Single-Turnover Assay

Cas9 (25 nM) was pre-incubated 15 min at 37° C. in cleavage buffer (20mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl₂, 0.5 mM DTT, 0.1 mM EDTA) withduplexed tracrRNA:crRNA-sp2 (25 nM, 1:1) or both RNAs (25 nM) notpreannealed and the reaction was started by adding protospacer 2 plasmidDNA (5 nM). The reaction mix was incubated at 37° C. At defined timeintervals, samples were withdrawn from the reaction, 5×SDS loadingbuffer (30% glycerol, 1.2% SDS, 250 mM EDTA) was added to stop thereaction and the cleavage was monitored by 1% agarose gelelectrophoresis and ethidium bromide staining. The same was done for thesingle turnover kinetics without pre-incubation of Cas9 and RNA, whereprotospacer 2 plasmid DNA (5 nM) was mixed in cleavage buffer withduplex tracrRNA:crRNA-sp2 (25 nM) or both RNAs (25 nM) not pre-annealed,and the reaction was started by addition of Cas9 (25 nM). Percentage ofcleavage was analyzed by densitometry and the average of threeindependent experiments was plotted against time. The data were fit bynonlinear regression analysis and the cleavage rates (k_(obs)[min⁻¹])were calculated.

Multiple-Turnover Assay

Cas9 (1 nM) was pre-incubated for 15 min at 37° C. in cleavage buffer(20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl₂, 0.5 mM DTT, 0.1 mM EDTA)with pre-annealed tracrRNA:crRNA-sp2 (1 nM, 1:1). The reaction wasstarted by addition of protospacer 2 plasmid DNA (5 nM). At defined timeintervals, samples were withdrawn and the reaction was stopped by adding5×SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA). The cleavagereaction was resolved by 1% agarose gel electrophoresis, stained withethidium bromide and the percentage of cleavage was analyzed bydensitometry. The results of four independent experiments were plottedagainst time (min).

Oligonucleotide DNA Cleavage Assay

DNA oligonucleotides (10 pmol) were radiolabeled by incubating with 5units T4 polynucleotide kinase (New England Biolabs) and ˜3-6 pmol(˜20-40 mCi) [γ-32P]-ATP (Promega) in 1×T4 polynucleotide kinasereaction buffer at 37° C. for 30 min, in a 50 μL reaction. After heatinactivation (65° C. for 20 min), reactions were purified through anIllustra MicroSpin G-25 column (GE Healthcare) to remove unincorporatedlabel. Duplex substrates (100 nM) were generated by annealing labeledoligonucleotides with equimolar amounts of unlabeled complementaryoligonucleotide at 95° C. for 3 min, followed by slow cooling to roomtemperature. For cleavage assays, tracrRNA and crRNA were annealed byheating to 95° C. for 30 s, followed by slow cooling to roomtemperature. Cas9 (500 nM final concentration) was pre-incubated withthe annealed tracrRNA:crRNA duplex (500 nM) in cleavage assay buffer (20mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, 5% glycerol) in atotal volume of 9 μl. Reactions were initiated by the addition of 1 μltarget DNA (10 nM) and incubated for 1 h at 37° C. Reactions werequenched by the addition of 20 μl of loading dye (5 mM EDTA, 0.025% SDS,5% glycerol in formamide) and heated to 95° C. for 5 min. Cleavageproducts were resolved on 12% denaturing polyacrylamide gels containing7 M urea and visualized by phosphorimaging (Storm, GE Life Sciences).Cleavage assays testing PAM requirements (FIG. 13B) were carried outusing DNA duplex substrates that had been pre-annealed and purified onan κ% native acrylamide gel, and subsequently radiolabeled at both 5′ends. The reactions were set-up and analyzed as above.

Electrophoretic Mobility Shift Assays

Target DNA duplexes were formed by mixing of each strand (10 nmol) indeionized water, heating to 95° C. for 3 min and slow cooling to roomtemperature. All DNAs were purified on 8% native gels containing 1×TBE.DNA bands were visualized by UV shadowing, excised, and eluted bysoaking gel pieces in DEPC-treated H₂O. Eluted DNA was ethanolprecipitated and dissolved in DEPC-treated H₂O. DNA samples were 5′ endlabeled with [γ-32P]-ATP using T4 polynucleotide kinase (New EnglandBiolabs) for 30 min at 37° C. PNK was heat denatured at 65° C. for 20min, and unincorporated radiolabel was removed using an IllustraMicroSpin G-25 column (GE Healthcare). Binding assays were performed inbuffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTTand 10% glycerol in a total volume of 10 μl. Cas9 D10A/H840A doublemutant was programmed with equimolar amounts of pre-annealedtracrRNA:crRNA duplex and titrated from 100 pM to 1 μM. Radiolabeled DNAwas added to a final concentration of 20 pM. Samples were incubated for1 h at 37° C. and resolved at 4° C. on an 8% native polyacrylamide gelcontaining 1×TBE and 5 mM MgCl₂. Gels were dried and DNA visualized byphosphorimaging.

In Silico Analysis of DNA and Protein Sequences

Vector NTI package (Invitrogen) was used for DNA sequence analysis(Vector NTI) and comparative sequence analysis of proteins (AlignX).

In Silico Modeling of RNA Structure and Co-Folding

In silico predictions were performed using the Vienna RNA packagealgorithms (42, 43). RNA secondary structures and co-folding models werepredicted with RNAfold and RNAcofold, respectively and visualized withVARNA (44).

Results

Bacteria and archaea have evolved RNA mediated adaptive defense systemscalled clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated (Cas) that protect organisms from invadingviruses and plasmids (1-3). We show that in a subset of these systems,the mature crRNA that is base-paired to trans-activating crRNA(tracrRNA) forms a two-RNA structure that directs the CRISPR-associatedprotein Cas9 to introduce double-stranded (ds) breaks in target DNA. Atsites complementary to the crRNA-guide sequence, the Cas9 HNH nucleasedomain cleaves the complementary strand, whereas the Cas9 RuvC-likedomain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA,when engineered as a single RNA chimera, also directs sequence-specificCas9 dsDNA cleavage. These studies reveal a family of endonucleases thatuse dual-RNAs for site-specific DNA cleavage and highlight the abilityto exploit the system for RNA-programmable genome editing.

CRISPR/Cas defense systems rely on small RNAs for sequence-specificdetection and silencing of foreign nucleic acids. CRISPR/Cas systems arecomposed of cas genes organized in operon(s) and CRISPR array(s)consisting of genome-targeting sequences (called spacers) interspersedwith identical repeats (1-3). CRISPR/Cas-mediated immunity occurs inthree steps. In the adaptive phase, bacteria and archaea harboring oneor more CRISPR loci respond to viral or plasmid challenge by integratingshort fragments of foreign sequence (protospacers) into the hostchromosome at the proximal end of the CRISPR array (1-3). In theexpression and interference phases, transcription of the repeat spacerelement into precursor CRISPR RNA (pre-crRNA) molecules followed byenzymatic cleavage yields the short crRNAs that can pair withcomplementary protospacer sequences of invading viral or plasmid targets(4-11). Target recognition by crRNAs directs the silencing of theforeign sequences by means of Cas proteins that function in complex withthe crRNAs (10, 12-20).

There are three types of CRISPR/Cas systems (21-23). The type I and IIIsystems share some overarching features: specialized Cas endonucleasesprocess the pre-crRNAs, and oncemature, each crRNA assembles into alarge multi-Cas protein complex capable of recognizing and cleavingnucleic acids complementary to the crRNA. In contrast, type II systemsprocess precrRNAs by a different mechanism in which a trans-activatingcrRNA (tracrRNA) complementary to the repeat sequences in pre-crRNAtriggers processing by the double-stranded (ds) RNAspecific ribonucleaseRNase III in the presence of the Cas9 (formerly Csn1) protein (FIG. 15)(4, 24). Cas9 is thought to be the sole protein responsible forcrRNA-guided silencing of foreign DNA (25-27).

We show that in type II systems, Cas9 proteins constitute a family ofenzymes that require a base-paired structure formed between theactivating tracrRNA and the targeting crRNA to cleave target dsDNA.Site-specific cleavage occurs at locations determined by bothbase-pairing complementarity between the crRNA and the targetprotospacer DNA and a short motif [referred to as the protospaceradjacent motif (PAM)] juxtaposed to the complementary region in thetarget DNA. Our study further demonstrates that the Cas9 endonucleasefamily can be programmed with single RNA molecules to cleave specificDNA sites, thereby facilitating the development of a simple andversatile RNA-directed system to generate dsDNA breaks for genometargeting and editing.

Cas9 is a DNA Endonuclease Guided by Two RNAs

Cas9, the hallmark protein of type II systems, has been hypothesized tobe involved in both crRNA maturation and crRNA-guided DNA interference(FIG. 15) (4, 25-27). Cas9 is involved in crRNA maturation (4), but itsdirect participation in target DNA destruction has not beeninvestigated. To test whether and how Cas9 might be capable of targetDNA cleavage, we used an overexpression system to purify Cas9 proteinderived from the pathogen Streptococcus pyogenes (FIG. 16A-16B, seesupplementary materials and methods) and tested its ability to cleave aplasmid DNA or an oligonucleotide duplex bearing a protospacer sequencecomplementary to a mature crRNA, and a bona fide PAM. We found thatmature crRNA alone was incapable of directing Cas9-catalyzed plasmid DNAcleavage (FIG. 10A and FIG. 17A). However, addition of tracrRNA, whichcan pair with the repeat sequence of crRNA and is essential to crRNAmaturation in this system, triggered Cas9 to cleave plasmidDNA (FIG. 10Aand FIG. 17A). The cleavage reaction required both magnesium and thepresence of a crRNA sequence complementary to the DNA; a crRNA capableof tracrRNAbase pairing but containing a noncognate target DNA-bindingsequence did not support Cas9-catalyzed plasmid cleavage (FIG. 10A; FIG.17A, compare crRNA-sp2 to crRNA-sp1; and FIG. 18A). We obtained similarresults with a short linear dsDNA substrate (FIG. 10B and FIGS. 17B and17C). Thus, the trans-activating tracrRNA is a small noncoding RNA withtwo critical functions: triggering pre-crRNA processing by the enzymeRNase III (4) and subsequently activating crRNA-guided DNA cleavage byCas9.

Cleavage of both plasmid and short linear dsDNA by tracrRNA:crRNA-guidedCas9 is site specific (FIGS. 10C to 10E, and FIGS. 19A and 19B). PlasmidDNA cleavage produced blunt ends at a position three base pairs upstreamof the PAM sequence (FIGS. 10C and 10E, and FIGS. 19A and 19C) (26).Similarly, within short dsDNA duplexes, the DNA strand that iscomplementary to the target-binding sequence in the crRNA (thecomplementary strand) is cleaved at a site three base pairs upstream ofthe PAM (FIGS. 10D and 10E, and FIGS. 19B and 19C). The noncomplementaryDNA strand is cleaved at one or more sites within three to eight basepairs upstream of the PAM. Further investigation revealed that thenoncomplementary strand is first cleaved endonucleolytically andsubsequently trimmed by a 3′-5′ exonuclease activity (FIG. 18B). Thecleavage rates by Cas9 under single-turnover conditions ranged from 0.3to 1 min-1, comparable to those of restriction endonucleases (FIG. 20A),whereas incubation of wildtype (WT) Cas9-tracrRNA:crRNA complex with afivefold molar excess of substrate DNA provided evidence that thedual-RNA-guided Cas9 is a multiple-turnover enzyme (FIG. 20B). Incontrast to the CRISPR type I Cascade complex (18), Cas9 cleaves bothlinearized and supercoiled plasmids (FIGS. 10A and 11A). Therefore, aninvading plasmid can, in principle, be cleaved multiple times by Cas9proteins programmed with different crRNAs.

(FIG. 10A) Cas9 was programmed with a 42-nucleotide crRNA-sp2 (crRNAcontaining a spacer 2 sequence) in the presence or absence of75-nucleotide tracrRNA. The complex was added to circular orXhoI-linearized plasmid DNA bearing a sequence complementary to spacer 2and a functional PAM. crRNA-sp1, specificity control; M, DNA marker;kbp, kilo-base pair. See FIG. 17A. (FIG. 10B) Cas9 was programmed withcrRNA-sp2 and tracrRNA (nucleotides 4 to 89). The complex was incubatedwith double- or single-stranded DNAs harboring a sequence complementaryto spacer 2 and a functional PAM (4). The complementary ornoncomplementary strands of the DNA were 5′-radiolabeled and annealedwith a nonlabeled partner strand. nt, nucleotides. See FIGS. 17B and17C. (FIG. 10C) Sequencing analysis of cleavage products from FIG. 10A.Termination of primer extension in the sequencing reaction indicates theposition of the cleavage site. The 3′ terminal A overhang (asterisks) isan artifact of the sequencing reaction. See FIGS. 19A and 19C. (FIG.10D) The cleavage products from FIG. 10B were analyzed alongside 5′end-labeled size markers derived from the complementary andnoncomplementary strands of the target DNA duplex. M, marker; P,cleavage product. See FIGS. 19B and 19C (FIG. 10E) Schematicrepresentation of tracrRNA, crRNA-sp2, and protospacer 2 DNA sequences.Regions of crRNA complementarity to tracrRNA (overline) and theprotospacer DNA (underline) are represented. The PAM sequence islabeled; cleavage sites mapped in (FIG. 10C) and (FIG. 10D) arerepresented by white-filled arrows (FIG. 10C), a black-filled arrow[(FIG. 10D), complementary strand], and a black bar [(FIG. 10D),noncomplementary strand].

FIG. 15 depicts the type II RNA-mediated CRISPR/Cas immune pathway. Theexpression and interference steps are represented in the drawing. Thetype II CRISPR/Cas loci are composed of an operon of four genes encodingthe proteins Cas9, Cas1, Cas2 and Csn2, a CRISPR array consisting of aleader sequence followed by identical repeats (black rectangles)interspersed with unique genome-targeting spacers (diamonds) and asequence encoding the trans-activating tracrRNA. Represented here is thetype II CRISPR/Cas locus of S. pyogenes SF370 (Accession numberNC_002737) (4). Experimentally confirmed promoters and transcriptionalterminator in this locus are indicated (4). The CRISPR array istranscribed as a precursor CRISPR RNA (pre-crRNA) molecule thatundergoes a maturation process specific to the type II systems (4). InS. pyogenes SF370, tracrRNA is transcribed as two primary transcripts of171 and 89 nt in length that have complementarity to each repeat of thepre-crRNA. The first processing event involves pairing of tracrRNA topre-crRNA, forming a duplex RNA that is recognized and cleaved by thehousekeeping endoribonuclease RNase III in the presence of the Cas9protein. RNase III-mediated cleavage of the duplex RNA generates a 75-ntprocessed tracrRNA and a 66-nt intermediate crRNAs consisting of acentral region containing a sequence of one spacer, flanked by portionsof the repeat sequence. A second processing event, mediated by unknownribonuclease(s), leads to the formation of mature crRNAs of 39 to 42 ntin length consisting of 5′-terminal spacer-derived guide sequence andrepeat-derived 3′-terminal sequence. Following the first and secondprocessing events, mature tracrRNA remains paired to the mature crRNAsand bound to the Cas9 protein. In this ternary complex, the dualtracrRNA:crRNA structure acts as guide RNA that directs the endonucleaseCas9 to the cognate target DNA. Target recognition by theCas9-tracrRNA:crRNA complex is initiated by scanning the invading DNAmolecule for homology between the protospacer sequence in the target DNAand the spacer-derived sequence in the crRNA. In addition to the DNAprotospacer-crRNA spacer complementarity, DNA targeting requires thepresence of a short motif (NGG, where N can be any nucleotide) adjacentto the protospacer (protospacer adjacent motif—PAM). Following pairingbetween the dual-RNA and the protospacer sequence, an R-loop is formedand Cas9 subsequently introduces a double-stranded break (DSB) in theDNA. Cleavage of target DNA by Cas9 requires two catalytic domains inthe protein. At a specific site relative to the PAM, the HNH domaincleaves the complementary strand of the DNA while the RuvC-like domaincleaves the noncomplementary strand.

(FIG. 16A) S. pyogenes Cas9 was expressed in E. coli as a fusion proteincontaining an N-terminal His6-MBP tag and purified by a combination ofaffinity, ion exchange and size exclusion chromatographic steps. Theaffinity tag was removed by TEV protease cleavage following the affinitypurification step. Shown is a chromatogram of the final size exclusionchromatography step on a Superdex 200 (16/60) column. Cas9 elutes as asingle monomeric peak devoid of contaminating nucleic acids, as judgedby the ratio of absorbances at 280 and 260 nm. Inset; eluted fractionswere resolved by SDS-PAGE on a 10% polyacrylamide gel and stained withSimplyBlue Safe Stain (Invitrogen). (FIG. 16B) SDS-PAGE analysis ofpurified Cas9 orthologs. Cas9 orthologs were purified as described inSupplementary Materials and Methods. 2.5 μg of each purified Cas9 wereanalyzed on a 4-20% gradient polyacrylamide gel and stained withSimplyBlue Safe Stain.

FIG. 17A-17C (also see FIG. 10A-10E). The protospacer 1 sequenceoriginates from S. pyogenes SF370 (M1) SPy_0700, target of S. pyogenesSF370 crRNAsp1 (4). Here, the protospacer 1 sequence was manipulated bychanging the PAM from a nonfunctional sequence (TTG) to a functional one(TGG). The protospacer 4 sequence originates from S. pyogenes MGAS10750(M4) MGAS10750_Spy1285, target of S. pyogenes SF370 crRNA-sp4 (4). (FIG.17A) Protospacer 1 plasmid DNA cleavage guided by cognate tracrRNA:crRNAduplexes. The cleavage products were resolved by agarose gelelectrophoresis and visualized by ethidium bromide staining. M, DNAmarker; fragment sizes in base pairs are indicated. (FIG. 17B)Protospacer 1 oligonucleotide DNA cleavage guided by cognatetracrRNA:crRNA-sp1 duplex. The cleavage products were resolved bydenaturating polyacrylamide gel electrophoresis and visualized byphosphorimaging. Fragment sizes in nucleotides are indicated. (FIG. 17C)Protospacer 4 oligonucleotide DNA cleavage guided by cognatetracrRNA:crRNA-sp4 duplex. The cleavage products were resolved bydenaturating polyacrylamide gel electrophoresis and visualized byphosphorimaging. Fragment sizes in nucleotides are indicated. (FIG. 17A,FIG. 17B, FIG. 17C) Experiments in FIG. 17A were performed as in FIG.10A; in FIG. 17B and in FIG. 17C as in FIG. 10B. (FIG. 17B, FIG. 17C) Aschematic of the tracrRNA:crRNAtarget DNA interaction is shown below.The regions of crRNA complementarity to tracrRNA and the protospacer DNAare overlined and underlined, respectively. The PAM sequence is labeled.

FIG. 18 (also see FIG. 10A-10E). (FIG. 18A) Protospacer 2 plasmid DNAwas incubated with Cas9 complexed with tracrRNA:crRNA-sp2 in thepresence of different concentrations of Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺, Co²⁺,Ni²⁺ or Cu²⁺. The cleavage products were resolved by agarose gelelectrophoresis and visualized by ethidium bromide staining. Plasmidforms are indicated. (FIG. 18B) A protospacer 4 oligonucleotide DNAduplex containing a PAM motif was annealed and gel-purified prior toradiolabeling at both 5′ ends. The duplex (10 nM final concentration)was incubated with Cas9 programmed with tracrRNA (nucleotides 23-89) andcrRNAsp4 (500 nM final concentration, 1:1). At indicated time points(min), 10 μl aliquots of the cleavage reaction were quenched withformamide buffer containing 0.025% SDS and 5 mM EDTA, and analyzed bydenaturing polyacrylamide gel electrophoresis as in FIG. 10B. Sizes innucleotides are indicated.

(FIG. 19A) Mapping of protospacer 1 plasmid DNA cleavage. Cleavageproducts from FIG. 17A were analyzed by sequencing as in FIG. 10C. Notethat the 3′ terminal A overhang (asterisk) is an artifact of thesequencing reaction. (FIG. 19B) Mapping of protospacer 4 oligonucleotideDNA cleavage. Cleavage products from FIG. 17C were analyzed bydenaturing polyacrylamide gel electrophoresis alongside 5′ endlabeledoligonucleotide size markers derived from the complementary andnoncomplementary strands of the protospacer 4 duplex DNA. M, marker; P,cleavage product. Lanes 1-2: complementary strand. Lanes 3-4:non-complementary strand. Fragment sizes in nucleotides are indicated.(FIG. 19C) Schematic representations of tracrRNA, crRNA-sp1 andprotospacer 1 DNA sequences (top) and tracrRNA, crRNAsp4 and protospacer4 DNA sequences (bottom). tracrRNA:crRNA forms a dual-RNA structuredirected to complementary protospacer DNA through crRNA-protospacer DNApairing. The regions of crRNA complementary to tracrRNA and theprotospacer DNA are overlined and underlined, respectively. The cleavagesites in the complementary and noncomplementary DNA strands mapped in(FIG. 19A) (top) and (FIG. 19B) (bottom) are represented with arrows(FIG. 19A and FIG. 19B, complementary strand) and a black bar (FIG. 19B,noncomplementary strand) above the sequences, respectively.

(FIG. 20A) Single turnover kinetics of Cas9 under different RNApre-annealing and protein-RNA pre-incubation conditions. Protospacer 2plasmid DNA was incubated with either Cas9 pre-incubated withpre-annealed tracrRNA:crRNA-sp2 (∘), Cas9 not pre-incubated withpre-annealed tracrRNA:crRNA-sp2 (⋅), Cas9 pre-incubated with notpre-annealed tracrRNA and crRNA-sp2 (□) or Cas9 not pre-incubated withnot pre-annealed RNAs (▪). The cleavage activity was monitored in atime-dependent manner and analyzed by agarose gel electrophoresisfollowed by ethidium bromide staining. The average percentage ofcleavage from three independent experiments is plotted against the time(min) and fitted with a nonlinear regression. The calculated cleavagerates (k_(obs)) are shown in the table. The results suggest that thebinding of Cas9 to the RNAs is not rate-limiting under the conditionstested. Plasmid forms are indicated. The obtained k_(obs) values arecomparable to those of restriction endonucleases which are typically ofthe order of 1-10 per min (45-47). (FIG. 20B) Cas9 is a multipleturnover endonuclease. Cas9 loaded with duplexed tracrRNA:crRNA-sp2 (1nM, 1:1:1—indicated with gray line on the graph) was incubated with a5-fold excess of native protospacer 2 plasmid DNA. Cleavage wasmonitored by withdrawing samples from the reaction at defined timeintervals (0 to 120 min) followed by agarose gel electrophoresisanalysis (top) and determination of cleavage product amount (nM)(bottom). Standard deviations of three independent experiments areindicated. In the time interval investigated, 1 nM Cas9 was able tocleave ˜2.5 nM plasmid DNA.

Each Cas9 Nuclease Domain Cleaves One DNA Strand

Cas9 contains domains homologous to both HNH and RuvC endonucleases(FIG. 11A and FIG. 3A and FIG. 3B) (21-23, 27, 28). We designed andpurified Cas9 variants containing inactivating point mutations in thecatalytic residues of either the HNH or RuvC-like domains (FIG. 11A andFIG. 3A and FIG. 3B) (23, 27). Incubation of these variant Cas9 proteinswith native plasmid DNA showed that dual-RNA-guided mutant Cas9 proteinsyielded nicked open circular plasmids, whereas the WT Cas9protein-tracrRNA:crRNA complex produced a linear DNA product (FIG. 10Aand FIG. 11A and FIG. 17A and FIG. 25A). This result indicates that theCas9 HNH and RuvC-like domains each cleave one plasmid DNA strand. Todetermine which strand of the target DNA is cleaved by each Cas9catalytic domain, we incubated the mutant Cas9-tracrRNA:crRNA complexeswith short dsDNA substrates in which either the complementary ornoncomplementary strand was radiolabeled at its 5′ end. The resultingcleavage products indicated that the Cas9 HNH domain cleaves thecomplementary DNA strand, whereas the Cas9 RuvC-like domain cleaves thenoncomplementaryDNAstrand (FIG. 11B and FIG. 21B).

(FIG. 11A) (Top) Schematic representation of Cas9 domain structureshowing the positions of domain mutations. D10A, Asp10→Ala10; H840A;His840→Ala840. Complexes of WT or nuclease mutant Cas9 proteins withtracrRNA:crRNA-sp2 were assayed for endonuclease activity as in FIG.10A. (FIG. 11B) Complexes of WT Cas9 or nuclease domain mutants withtracrRNA and crRNA-sp2 were tested for activity as in FIG. 10B.

FIG. 3A and FIG. 3B The amino-acid sequence of Cas9 from S. pyogenes(SEQ ID NO:8) is represented. Cas9/Csn1 proteins from various diversespecies have 2 domains that include motifs homologous to both HNH andRuvC endonucleases. (FIG. 3A) Motifs 1-4 (motif numbers are marked onleft side of sequence) are shown for S. pyogenes Cas9/Csn1. The threepredicted RuvC-like motifs (1, 2, 4) and the predicted HNH motif (3) areoverlined. Residues Asp10 and His840, which were substituted by Ala inthis study are highlighted by an asterisk above the sequence. Underlinedresidues are highly conserved among Cas9 proteins from differentspecies. Mutations in underlined residues are likely to have functionalconsequences on Cas9 activity. Note that in the present study couplingof the two nuclease-like activities is experimentally demonstrated (FIG.11A-11B and FIG. 21A-21B). (FIG. 3B) Domains 1 (amino acids 7-166) and 2(amino acids 731-1003), which include motifs 1-4, are depicted for S.pyogenes Cas9/Csn1. Refer to Table land FIG. 5 for additionalinformation.

FIG. 21A-21B Protospacer DNA cleavage by cognate tracrRNA:crRNA-directedCas9 mutants containing mutations in the HNH or RuvC-like domain. (FIG.21A) Protospacer 1 plasmid DNA cleavage. The experiment was performed asin FIG. 1l A. Plasmid DNA conformations and sizes in base pairs areindicated. (FIG. 21B) Protospacer 4 oligonucleotide DNA cleavage. Theexperiment was performed as in FIG. 11B. Sizes in nucleotides areindicated.

Dual-RNA Requirements for Target DNA Binding and Cleavage

tracrRNA might be required for targetDNA binding and/or to stimulate thenuclease activity of Cas9 downstream of target recognition. Todistinguish between these possibilities, we used an electrophoreticmobility shift assay to monitor target DNA binding by catalyticallyinactive Cas9 in the presence or absence of crRNA and/or tracrRNA.Addition of tracrRNA substantially enhanced target DNA binding by Cas9,whereas we observed little specific DNA binding with Cas9 alone orCas9-crRNA (FIG. 22). This indicates that tracrRNA is required fortarget DNA recognition, possibly by properly orienting the crRNA forinteraction with the complementary strand of target DNA. The predictedtracrRNA:crRNA secondary structure includes base pairing between the 22nucleotides at the 3′ terminus of the crRNA and a segment near the 5′end of the mature tracrRNA (FIG. 10E). This interaction creates astructure in which the 5′-terminal 20 nucleotides of the crRNA, whichvary in sequence in different crRNAs, are available for target DNAbinding. The bulk of the tracrRNA downstream of the crRNA basepairingregion is free to form additional RNA structure(s) and/or to interactwith Cas9 or the target DNA site. To determine whether the entire lengthof the tracrRNA is necessary for site specific Cas9-catalyzed DNAcleavage, we tested Cas9-tracrRNA:crRNA complexes reconstituted usingfull-length mature (42-nucleotide) crRNA and various truncated forms oftracrRNA lacking sequences at their 5′ or 3′ ends. These complexes weretested for cleavage using a short target dsDNA. A substantiallytruncated version of the tracrRNA retaining nucleotides 23 to 48 of thenative sequence was capable of supporting robust dual-RNA-guidedCas9-catalyzed DNA cleavage (FIG. 12A and FIG. 12C, and FIG. 23A andFIG. 23B). Truncation of the crRNA from either end showed thatCas9-catalyzed cleavage in the presence of tracrRNA could be triggeredwith crRNAs missing the 3′-terminal 10 nucleotides (FIG. 12B and FIG.12C). In contrast, a 10-nucleotide deletion from the 5′ end of crRNAabolished DNA cleavage by Cas9 (FIG. 12B). We also analyzed Cas9orthologs from various bacterial species for their ability to support S.pyogenes tracrRNA:crRNA-guided DNA cleavage. In contrast to closelyrelated S. pyogenes Cas9 orthologs, more distantly related orthologswere not functional in the cleavage reaction (FIG. 24A-24D). Similarly,S. pyogenes Cas9 guided by tracrRNA:crRNA duplexes originating from moredistant systems was unable to cleave DNA efficiently (FIG. 24A-24D).Species specificity of dual-RNA-guided cleavage of DNA indicatescoevolution of Cas9, tracrRNA, and the crRNA repeat, as well as theexistence of a still unknown structure and/or sequence in the dual-RNAthat is critical for the formation of the ternary complex with specificCas9 orthologs.

To investigate the protospacer sequence requirements for type IICRISPR/Cas immunity in bacterial cells, we analyzed a series ofprotospacer-containing plasmid DNAs harboring single-nucleotidemutations for their maintenance following transformation in S. pyogenesand their ability to be cleaved by Cas9 in vitro. In contrast to pointmutations introduced at the 5′ end of the protospacer, mutations in theregion close to the PAM and the Cas9 cleavage sites were not toleratedin vivo and resulted in decreased plasmid cleavage efficiency in vitro(FIG. 12D). Our results are in agreement with a previous report ofprotospacer escape mutants selected in the type II CRISPR system from S.thermophilus in vivo (27, 29). Furthermore, the plasmid maintenance andcleavage results hint at the existence of a “seed” region located at the3′ end of the protospacer sequence that is crucial for the interactionwith crRNA and subsequent cleavage by Cas9. In support of this notion,Cas9 enhanced complementary DNA strand hybridization to the crRNA; thisenhancement was the strongest in the 3′-terminal region of the crRNAtargeting sequence (FIG. 25A-25C). Corroborating this finding, acontiguous stretch of at least 13 base pairs between the crRNA and thetarget DNA site proximal to the PAM is required for efficient targetcleavage, whereas up to six contiguous mismatches in the 5′-terminalregion of the protospacer are tolerated (FIG. 12E). These findings arereminiscent of the previously observed seed-sequence requirements fortarget nucleic acid recognition in Argonaute proteins (30, 31) and theCascade and Csy CRISPR complexes (13, 14).

(FIG. 12A) Cas9-tracrRNA: crRNA complexes were reconstituted using42-nucleotide crRNA-sp2 and truncated tracrRNA constructs and wereassayed for cleavage activity as in FIG. 10B. (FIG. 12B) Cas9 programmedwith full-length tracrRNA and crRNA-sp2 truncations was assayed foractivity as in (FIG. 12A). (FIG. 12C) Minimal regions of tracrRNA andcrRNA capable of guiding Cas9-mediated DNA cleavage (shaded region).(FIG. 12D) Plasmids containing WT or mutant protospacer 2 sequences withindicated point mutations were cleaved in vitro by programmed Cas9 as inFIG. 10A and used for transformation assays of WT or pre-crRNA-deficientS. pyogenes. The transformation efficiency was calculated ascolony-forming units (CFU) per microgram of plasmid DNA. Error barsrepresent SDs for three biological replicates. (FIG. 12E) Plasmidscontaining WT and mutant protospacer 2 inserts with varying extent ofcrRNA-target DNA mismatches (bottom) were cleaved in vitro by programmedCas9 (top). The cleavage reactions were further digested with XmnI. The1880- and 800-bp fragments are Cas9-generated cleavage products. M, DNAmarker.

FIG. 22 Electrophoretic mobility shift assays were performed usingprotospacer 4 target DNA duplex and Cas9 (containing nuclease domaininactivating mutations D10A and H840) alone or in the presence ofcrRNA-sp4, tracrRNA (75 nt), or both. The target DNA duplex wasradiolabeled at both 5′ ends. Cas9 (D10/H840A) and complexes weretitrated from 1 nM to 1 M. Binding was analyzed by 8% nativepolyacrylamide gel electrophoresis and visualized by phosphorimaging.Note that Cas9 alone binds target DNA with moderate affinity. Thisbinding is unaffected by the addition of crRNA, suggesting that thisrepresents sequence nonspecific interaction with the dsDNA. Furthermore,this interaction can be outcompeted by tracrRNA alone in the absence ofcrRNA. In the presence of both crRNA and tracrRNA, target DNA binding issubstantially enhanced and yields a species with distinctelectrophoretic mobility, indicative of specific target DNA recognition.

FIG. 23A-23B A fragment of tracrRNA encompassing a part of the crRNApaired region and a portion of the downstream region is sufficient todirect cleavage of protospacer oligonucleotide DNA by Cas9. (FIG. 23A)Protospacer 1 oligonucleotide DNA cleavage and (FIG. 23B) Protospacer 4oligonucleotide DNA cleavage by Cas9 guided with a mature cognate crRNAand various tracrRNA fragments. (FIG. 23A, FIG. 23B) Sizes innucleotides are indicated.

FIG. 24A-24D Like Cas9 from S. pyogenes, the closely related Cas9orthologs from the Gram-positive bacteria L. innocua and S. thermophiluscleave protospacer DNA when targeted by tracrRNA:crRNA from S. pyogenes.However, under the same conditions, DNA cleavage by the less closelyrelated Cas9 orthologs from the Gramnegative bacteria C. jejuni and N.meningitidis is not observed. Spy, S. pyogenes SF370 (Accession NumberNC_002737); Sth, S. thermophilus LMD-9 (STER_1477 Cas9 ortholog;Accession Number NC_008532); Lin, L. innocua Clip11262 (Accession NumberNC_003212); Cje, C. jejuni NCTC 11168 (Accession Number NC_002163); Nme,N. meningitidis A Z2491 (Accession Number NC_003116). (FIG. 24A)Cleavage of protospacer plasmid DNA. Protospacer 2 plasmid DNA (300 ng)was subjected to cleavage by different Cas9 orthologs (500 nM) guided byhybrid tracrRNA:crRNA-sp2 duplexes (500 nM, 1:1) from different species.To design the RNA duplexes, we predicted tracrRNA sequences from L.innocua and N. meningitidis based on previously published Northern blotdata (4). The dual-hybrid RNA duplexes consist of species specifictracrRNA and a heterologous crRNA. The heterologous crRNA sequence wasengineered to contain S. pyogenes DNA-targeting sp2 sequence at the 5′end fused to L. innocua or N. meningitidis tracrRNA-binding repeatsequence at the 3′ end. Cas9 orthologs from S. thermophilus and L.innocua, but not from N. meningitidis or C. jejuni, can be guided by S.pyogenes tracrRNA:crRNA-sp2 to cleave protospacer 2 plasmid DNA, albeitwith slightly decreased efficiency. Similarly, the hybrid L. innocuatracrRNA:crRNA-sp2 can guide S. pyogenes Cas9 to cleave the target DNAwith high efficiency, whereas the hybrid N. meningitidistracrRNA:crRNA-sp2 triggers only slight DNA cleavage activity by S.pyogenes Cas9. As controls, N. meningitidis and L. innocua Cas9orthologs cleave protospacer 2 plasmid DNA when guided by the cognatehybrid tracrRNA:crRNA-sp2. Note that as mentioned above, the tracrRNAsequence of N. meningitidis is predicted only and has not yet beenconfirmed by RNA sequencing. Therefore, the low efficiency of cleavagecould be the result of either low activity of the Cas9 orthologs or theuse of a nonoptimally designed tracrRNA sequence. (FIG. 24B) Cleavage ofprotospacer oligonucleotide DNA. 5′-end radioactively labeledcomplementary strand oligonucleotide (10 nM) pre-annealed with unlabelednoncomplementary strand oligonucleotide (protospacer 1) (10 nM) (left)or 5′-end radioactively labeled noncomplementary strand oligonucleotide(10 nM) pre-annealed with unlabeled complementary strand oligonucleotide(10 nM) (right) (protospacer 1) was subjected to cleavage by variousCas9 orthologs (500 nM) guided by tracrRNA:crRNA-sp1 duplex from S.pyogenes (500 nM, 1:1). Cas9 orthologs from S. thermophilus and L.innocua, but not from N. meningitidis or C. jejuni can be guided by S.pyogenes cognate dual-RNA to cleave the protospacer oligonucleotide DNA,albeit with decreased efficiency. Note that the cleavage site on thecomplementary DNA strand is identical for all three orthologs. Cleavageof the noncomplementary strand occurs at distinct positions. (FIG. 24C)Amino acid sequence identity of Cas9 orthologs. S. pyogenes, S.thermophilus and L. innocua Cas9 orthologs share high percentage ofamino acid identity. In contrast, the C. jejuni and N. meningitidis Cas9proteins differ in sequence and length (˜300-400 amino acids shorter).(FIG. 24D) Co-foldings of engineered species-specific heterologous crRNAsequences with the corresponding tracrRNA orthologs from S. pyogenes(experimentally confirmed, (4)), L. innocua (predicted) or N.meningitidis (predicted). tracrRNAs; crRNA spacer 2 fragments; and crRNArepeat fragments are traced and labeled. L. innocua and S. pyogeneshybrid tracrRNA:crRNA-sp2 duplexes share very similar structuralcharacteristics, albeit distinct from the N. meningitidis hybridtracrRNA:crRNA. Together with the cleavage data described above in FIG.24A and FIG. 24B, the co-folding predictions would indicate that thespecies-specificity cleavage of target DNA by Cas9-tracrRNA:crRNA isdictated by a still unknown structural feature in the tracrRNA:crRNAduplex that is recognized specifically by a cognate Cas9 ortholog. Itwas predicted that the species-specificity of cleavage observed in FIG.24A and FIG. 24B occurs at the level of binding of Cas9 todual-tracrRNA:crRNA. Dual-RNA guided Cas9 cleavage of target DNA can bespecies specific. Depending on the degree of diversity/evolution amongCas9 proteins and tracrRNA:crRNA duplexes, Cas9 and dual-RNA orthologsare partially interchangeable.

FIG. 25A-25C A series of 8-nucleotide DNA probes complementary toregions in the crRNA encompassing the DNA-targeting region andtracrRNA-binding region were analyzed for their ability to hybridize tothe crRNA in the context of a tracrRNA:crRNA duplex and theCas9-tracrRNA:crRNA ternary complex. (FIG. 25A) Schematic representationof the sequences of DNA probes used in the assay and their binding sitesin crRNA-sp4. (FIG. 25B-FIG. 25C) Electrophoretic mobility shift assaysof target DNA probes with tracrRNA:crRNA-sp4 or Cas9-tracrRNA:crRNA-sp4.The tracrRNA (15-89) construct was used in the experiment. Binding ofthe duplexes or complexes to target oligonucleotide DNAs was analyzed ona 16% native polyacrylamide gel and visualized by phosphorimaging.

A Short Sequence Motif Dictates R-Loop Formation

In multiple CRISPR/Cas systems, recognition of self versus nonself hasbeen shown to involve a short sequence motif that is preserved in theforeign genome, referred to as the PAM (27, 29, 32-34). PAMmotifs areonly a few base pairs in length, and their precise sequence and positionvary according to the CRISPR/Cas system type (32). In the S. pyogenestype II system, the PAM conforms to an NGG consensus sequence,containing two G:C base pairs that occur one base pair downstream of thecrRNA binding sequence, within the target DNA (4). Transformation assaysdemonstrated that the GG motif is essential for protospacer plasmid DNAelimination by CRISPR/Cas in bacterial cells (FIG. 26A), consistent withprevious observations in S. thermophilus (27). The motif is alsoessential for in vitro protospacer plasmid cleavage bytracrRNA:crRNA-guided Cas9 (FIG. 26B). To determine the role of the PAMin target DNA cleavage by the Cas9-tracrRNA: crRNA complex, we tested aseries of dsDNA duplexes containing mutations in the PAM sequence on thecomplementary or noncomplementary strands, or both (FIG. 13A). Cleavageassays using these substrates showed that Cas9-catalyzed DNA cleavagewas particularly sensitive to mutations in the PAM sequence on thenoncomplementary strand of the DNA, in contrast to complementary strandPAM recognition by type I CRISPR/Cas systems (18, 34). Cleavage oftarget single-stranded DNAs was unaffected by mutations of the PAMmotif. This observation suggests that the PAM motif is required only inthe context of target dsDNA and may thus be required to license duplexunwinding, strand invasion, and the formation of an R-loop structure.When we used a different crRNA-target DNA pair (crRNA-sp4 andprotospacer 4 DNA), selected due to the presence of a canonical PAM notpresent in the protospacer 2 target DNA, we found that both Gnucleotides of the PAM were required for efficient Cas9-catalyzed DNAcleavage (FIG. 13B and FIG. 26C). To determine whether the PAM plays adirect role in recruiting the Cas9-tracrRNA:crRNA complex to the correcttarget DNA site, we analyzed binding affinities of the complex fortarget DNA sequences by native gel mobility shift assays (FIG. 13C).Mutation of either G in the PAM sequence substantially reduced theaffinity of Cas9-tracrRNA: crRNA for the target DNA. This findingillustrates a role for the PAM sequence in target DNA binding by Cas9.

(FIG. 13A) Dual RNA-programmed Cas9 was tested for activity as in FIG.10B. WT and mutant PAM sequences in target DNAs are indicated withlines. (FIG. 13B) Protospacer 4 target DNA duplexes (labeled at both 5′ends) containing WT and mutant PAM motifs were incubated with Cas9programmed with tracrRNA:crRNA-sp4 (nucleotides 23 to 89). At theindicated time points (in minutes), aliquots of the cleavage reactionwere taken and analyzed as in FIG. 10B. (FIG. 13C) Electrophoreticmobility shift assays were performed using RNA-programmed Cas9(D10A/H840A) and protospacer 4 target DNA duplexes [same as in FIG. 13B]containing WT and mutated PAM motifs. The Cas9 (D10A/H840A)-RNA complexwas titrated from 100 pM to 1 mM.

(FIG. 26A) Mutations of the PAM sequence in protospacer 2 plasmid DNAabolish interference of plasmid maintenance by the Type II CRISPR/Cassystem in bacterial cells. Wild-type protospacer 2 plasmids with afunctional or mutated PAM were transformed into wild-type (strain SF370,also named EC904) and pre-crRNA-deficient mutant (EC1479) S. pyogenes asin FIG. 12D. PAM mutations are not tolerated by the Type II CRISPR/Cassystem in vivo. The mean values and standard deviations of threebiological replicates are shown. (FIG. 26B) Mutations of the PAMsequence in protospacer plasmid DNA abolishes cleavage byCas9-tracrRNA:crRNA. Wild type protospacer 2 plasmid with a functionalor mutated PAM were subjected to Cas9 cleavage as in FIG. 10A. The PAMmutant plasmids are not cleaved by the Cas9-tracrRNA:crRNA complex.(FIG. 26C) Mutations of the canonical PAM sequence abolish interferenceof plasmid maintenance by the Type II CRISPR/Cas system in bacterialcells. Wild-type protospacer 4 plasmids with a functional or mutated PAMwere cleaved with Cas9 programmed with tracrRNA and crRNA-sp2. Thecleavage reactions were carried out in the presence of the XmnIrestriction endonuclease to visualize the Cas9 cleavage products as twofragments (˜1880 and ˜800 bp). Fragment sizes in base pairs areindicated.

Cas9 can be Programmed with a Single Chimeric RNA

Examination of the likely secondary structure of the tracrRNA:crRNAduplex (FIGS. 10E and 12C) suggested the possibility that the featuresrequired for site-specific Cas9-catalyzed DNA cleavage could be capturedin a single chimeric RNA. Although the tracrRNA:crRNA target-selectionmechanism works efficiently in nature, the possibility of a singleRNA-guided Cas9 is appealing due to its potential utility for programmedDNA cleavage and genome editing (FIG. 1A-1B). We designed two versionsof a chimeric RNA containing a target recognition sequence at the 5′ endfollowed by a hairpin structure retaining the base-pairing interactionsthat occur between the tracrRNA and the crRNA (FIG. 14A). This singletranscript effectively fuses the 3′ end of crRNA to the 5′ end oftracrRNA, thereby mimicking the dual-RNA structure required to guidesite-specific DNA cleavage by Cas9. In cleavage assays using plasmidDNA, we observed that the longer chimeric RNA was able to guideCas9-catalyzed DNA cleavage in a manner similar to that observed for thetruncated tracrRNA:crRNA duplex (FIG. 14A and FIG. 27A and FIG. 27C).The shorter chimeric RNA did not work efficiently in this assay,confirming that nucleotides that are 5 to 12 positions beyond thetracrRNA:crRNA base-pairing interaction are important for efficient Cas9binding and/or target recognition. We obtained similar results incleavage assays using short dsDNA as a substrate, further indicatingthat the position of the cleavage site in target DNA is identical tothat observed using the dual tracrRNA:crRNA as a guide (FIG. 14B andFIG. 27B and FIG. 27C). Finally, to establish whether the design ofchimeric RNA might be universally applicable, we engineered fivedifferent chimeric guide RNAs to target a portion of the gene encodingthe green-fluorescent protein (GFP) (FIG. 28A to 28C) and tested theirefficacy against a plasmid carrying the GFP coding sequence in vitro. Inall five cases, Cas9 programmed with these chimeric RNAs efficientlycleaved the plasmid at the correct target site (FIG. 14C and FIG. 28D),indicating that rational design of chimeric RNAs is robust and could, inprinciple, enable targeting of any DNA sequence of interest with fewconstraints beyond the presence of a GG dinucleotide adjacent to thetargeted sequence.

FIG. 1A-1B A DNA-targeting RNA comprises a single stranded“DNA-targeting segment” and a “protein-binding segment,” which comprisesa stretch of double stranded RNA. (FIG. 1A) A DNA-targeting RNA cancomprise two separate RNA molecules (referred to as a “double-molecule”or “two-molecule” DNA-targeting RNA). A double-molecule DNA-targetingRNA comprises a “targeter-RNA” and an “activator-RNA.” (FIG. 1B) ADNA-targeting RNA can comprise a single RNA molecule (referred to as a“single-molecule” DNA-targeting RNA). A single-molecule DNA-targetingRNA comprises “linker nucleotides.”

(FIG. 14A) A plasmid harboring protospacer 4 target sequence and a WTPAM was subjected to cleavage by Cas9 programmed with tracrRNA(4-89):crRNA-sp4 duplex or in vitro-transcribed chimeric RNAsconstructed by joining the 3′ end of crRNA to the 5′ end of tracrRNAwith a GAAA tetraloop. Cleavage reactions were analyzed by restrictionmapping with XmnI. Sequences of chimeric RNAs A and B are shown withDNA-targeting (underline), crRNA repeat-derived sequences (overlined),and tracrRNA-derived (dashed underlined) sequences. (FIG. 14B)Protospacer 4 DNA duplex cleavage reactions were performed as in FIG.10B. (FIG. 14C) Five chimeric RNAs designed to target the GFP gene wereused to program Cas9 to cleave a GFP gene-containing plasmid. Plasmidcleavage reactions were performed as in FIG. 12E, except that theplasmid DNA was restriction mapped with AvrII after Cas9 cleavage.

(FIG. 27A) A single chimeric RNA guides Cas9-catalyzed cleavage ofcognate protospacer plasmid DNA (protospacer 1 and protospacer 2). Thecleavage reactions were carried out in the presence of the XmnIrestriction endonuclease to visualize the Cas9 cleavage products as twofragments (˜1880 and ˜800 bp). Fragment sizes in base pairs areindicated. (FIG. 27B) A single chimeric RNA guides Cas9-catalyzedcleavage of cognate protospacer oligonucleotide DNA (protospacer 1 andprotospacer 2). Fragment sizes in nucleotides are indicated. (FIG. 27C)Schematic representations of the chimeric RNAs used in the experiment.Sequences of chimeric RNAs A and B are shown with the 5′ protospacerDNA-targeting sequence of crRNA (underlined), the tracrRNA-bindingsequence of crRNA (overlined) and tracrRNA-derived sequence (dashedunderlined).

(FIG. 28A) Schematic representation of the GFP expression plasmidpCFJ127. The targeted portion of the GFP open reading frame is indicatedwith a black arrowhead. (FIG. 28B) Close-up of the sequence of thetargeted region. Sequences targeted by the chimeric RNAs are shown withgray bars. PAM dinucleotides are boxed. A unique SalI restriction siteis located 60 bp upstream of the target locus. (FIG. 28C) Left: TargetDNA sequences are shown together with their adjacent PAM motifs. Right:Sequences of the chimeric guide RNAs. (FIG. 28D) pCFJ127 was cleaved byCas9 programmed with chimeric RNAs GFP1-5, as indicated. The plasmid wasadditionally digested with SalI and the reactions were analyzed byelectrophoresis on a 3% agarose gel and visualized by staining with SYBRSafe.

CONCLUSIONS

A DNA interference mechanism was identified, involving a dual-RNAstructure that directs a Cas9 endonuclease to introduce site-specificdouble-stranded breaks in target DNA. The tracrRNA:crRNA-guided Cas9protein makes use of distinct endonuclease domains (HNH and RuvC-likedomains) to cleave the two strands in the target DNA. Target recognitionby Cas9 requires both a seed sequence in the crRNA and a GGdinucleotide-containing PAM sequence adjacent to the crRNA-bindingregion in the DNA target. We further show that the Cas9 endonuclease canbe programmed with guide RNA engineered as a single transcript to targetand cleave any dsDNA sequence of interest. The system is efficient,versatile, and programmable by changing the DNA target-binding sequencein the guide chimeric RNA. Zinc-finger nucleases andtranscription-activator-like effector nucleases have attractedconsiderable interest as artificial enzymes engineered to manipulategenomes (35-38). This represents alternative methodology based onRNA-programmed Cas9 that facilitates gene-targeting and genome-editingapplications.

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Example 2: RNA-Programmed Genome Editing in Human Cells

Data provided below demonstrate that Cas9 can be expressed and localizedto the nucleus of human cells, and that it assembles with single-guideRNA (“sgRNA”; encompassing the features required for both Cas9 bindingand DNA target site recognition) in a human cell. These complexes cangenerate double stranded breaks and stimulate non-homologous end joining(NHEJ) repair in genomic DNA at a site complementary to the sgRNAsequence, an activity that requires both Cas9 and the sgRNA. Extensionof the RNA sequence at its 3′ end enhances DNA targeting activity inliving cells. Further, experiments using extracts from transfected cellsshow that sgRNA assembly into Cas9 is the limiting factor forCas9-mediated DNA cleavage. These results demonstrate thatRNA-programmed genome editing works in living cells and in vivo.

Materials and Methods

Plasmid Design and Construction

The sequence encoding Streptococcus pyogenes Cas9 (residues 1-1368)fused to an HA epitope (amino acid sequence DAYPYDVPDYASL (SEQ IDNO:274)), a nuclear localization signal (amino acid sequencePKKKRKVEDPKKKRKVD (SEQ ID NO:275)) was codon optimized for humanexpression and synthesized by GeneArt. The DNA sequence is SEQ ID NO:276and the protein sequence is SEQ ID NO:277. Ligation-independent cloning(LIC) was used to insert this sequence into a pcDNA3.1-derived GFP andmCherry LIC vectors (vectors 6D and 6B, respectively, obtained from theUC Berkeley MacroLab), resulting in a Cas9-HA-NLS-GFP andCas9-HA-NLS-mCherry fusions expressed under the control of the CMVpromoter. Guide sgRNAs were expressed using expression vector pSilencer2.1-U6 puro (Life Technologies) and pSuper (Oligoengine). RNA expressionconstructs were generated by annealing complementary oligonucleotides toform the RNA-coding DNA sequence and ligating the annealed DNA fragmentbetween the BamHI and HindIII sites in pSilencer 2.1-U6 puro and BglIIand HindIII sites in pSuper.

Cell Culture Conditions and DNA Transfections

HEK293T cells were maintained in Dulbecco's modified eagle medium (DMEM)supplemented with 10% fetal bovine serum (FBS) in a 37° C. humidifiedincubator with 5% CO₂. Cells were transiently transfected with plasmidDNA using either X-tremeGENE DNA Transfection Reagent (Roche) orTurbofect Transfection Reagent (Thermo Scientific) with recommendedprotocols. Briefly, HEK293T cells were transfected at 60-80% confluencyin 6-well plates using 0.5 μg of the Cas9 expression plasmid and 2.0 μgof the RNA expression plasmid. The transfection efficiencies wereestimated to be 30-50% for Tubofect (FIG. 29E and FIG. 37A-37B) and80-90% for X-tremegene (FIG. 31B), based on the fraction of GFP-positivecells observed by fluorescence microscopy. 48 hours post transfection,cells were washed with phosphate buffered saline (PBS) and lysed byapplying 250 μl lysis buffer (20 mM Hepes pH 7.5, 100 mM potassiumchloride (KCl), 5 mM magnesium chloride (MgCl₂), 1 mM dithiothreitol(DTT), 5% glycerol, 0.1% Triton X-100, supplemented with Roche ProteaseInhibitor cocktail) and then rocked for 10 min at 4° C. The resultingcell lysate was divided into aliquots for further analysis. Genomic DNAwas isolated from 200 μl cell lysate using the DNeasy Blood and TissueKit (Qiagen) according to the manufacturer's protocol.

Western Blot Analysis of Cas9 Expression

HEK293T, transfected with the Cas9-HA-NLS-GFP expression plasmid, wereharvested and lysed 48 hours post transfection as above. 5 ul of lysatewere eletrophoresed on a 10% SDS polyacrylamide gel, blotter onto a PVDFmembrane and probed with HRP-conjugated anti-HA antibody (Sigma, 1:1000dilution in 1×PBS).

Surveyor Assay

The Surveyor assay was performed as previously described [10,12,13].Briefly, the human clathrin light chain A (CLTA) locus was PCR amplifiedfrom 200 ng of genomic DNA using a high fidelity polymerase, HerculaseII Fusion DNA Polymerase (Agilent Technologies) and forward primer5′-GCAGCAGAAGAAGCCTTTGT-3′ (SEQ ID NO: 1353) and reverse primer5′-TTCCTCCTCTCCCTCCTCTC-3′ (SEQ ID NO: 1354). 300 ng of the 360 bpamplicon was then denatured by heating to 95° C. and slowly reannealedusing a heat block to randomly rehybridize wild type and mutant DNAstrands. Samples were then incubated with Cel-1 nuclease (Surveyor Kit,Transgenomic) for 1 hour at 42° C. Cel-1 recognizes and cleaves DNAhelices containing mismatches (wild type:mutant hybridization). Cel-1nuclease digestion products were separated on a 10% acrylamide gel andvisualized by staining with SYBR Safe (Life Technologies).Quantification of cleavage bands was performed using ImageLab software(Bio-Rad). The percent cleavage was determined by dividing the averageintensity of cleavage products (160-200 bps) by the sum of theintensities of the uncleaved PCR product (360 bp) and the cleavageproduct.

In Vitro Transcription

Guide RNA was in vitro transcribed using recombinant T7 RNA polymeraseand a DNA template generated by annealing complementary syntheticoligonucleotides as previously described [14]. RNAs were purified byelectrophoresis on 7M urea denaturing acrylamide gel, ethanolprecipitated, and dissolved in DEPC-treated water.

Northern Blot Analysis

RNA was purified from HEK293T cells using the mirVana small-RNAisolation kit (Ambion). For each sample, 800 ng of RNA were separated ona 10% urea-PAGE gel after denaturation for 10 min at 70° C. in RNAloading buffer (0.5×TBE (pH7.5), 0.5 mg/ml bromophenol blue, 0.5 mgxylene cyanol and 47% formamide). After electrophoresis at 10 W in0.5×TBE buffer until the bromophenol blue dye reached the bottom of thegel, samples were electroblotted onto a Nytran membrane at 20 volts for1.5 hours in 0.5×TBE. The transferred RNAs were cross-linked onto theNytran membrane in UV-Crosslinker (Strategene) and were pre-hybridizedat 45° C. for 3 hours in a buffer containing 40% formamide, 5×SSC, 3XDernhardt's (0.1% each of ficoll, polyvinylpyrollidone, and BSA) and 200μg/ml Salmon sperm DNA. The pre-hybridized membranes were incubatedovernight in the prehybridization buffer supplemented with5′-³²P-labeled antisense DNA oligo probe at 1 million cpm/ml. Afterseveral washes in SSC buffer (final wash in 0.2×SCC), the membranes wereimaged phosphorimaging.

In Vitro Cleavage Assay

Cell lysates were prepared as described above and incubated withCLTA-RFP donor plasmid [10]. Cleavage reactions were carried out in atotal volume of 20 μl and contained 10 μl lysate, 2 μl of 5× cleavagebuffer (100 mM HEPES pH 7.5, 500 mM KCl, 25 mM MgCl₂, 5 mM DTT, 25%glycerol) and 300 ng plasmid. Where indicated, reactions weresupplemented with 10 pmol of in vitro transcribed CLTA1 sgRNA. Reactionswere incubated at 37° C. for one hour and subsequently digested with 10U of XhoI (NEB) for an additional 30 min at 37° C. The reactions werestopped by the addition of Proteinase K (Thermo Scientific) andincubated at 37° C. for 15 min. Cleavage products were analyzed byelectrophoresis on a 1% agarose gel and stained with SYBR Safe. Thepresence of ˜2230 and ˜3100 bp fragments is indicative of Cas9-mediatedcleavage.

Results

To test whether Cas9 could be programmed to cleave genomic DNA in livingcells, Cas9 was co-expressed together with an sgRNA designed to targetthe human clathrin light chain (CLTA) gene. The CLTA genomic locus haspreviously been targeted and edited using ZFNs [10]. We first tested theexpression of a human-codon-optimized version of the Streptococcuspyogenes Cas9 protein and sgRNA in human HEK293T cells. The 160 kDa Cas9protein was expressed as a fusion protein bearing an HA epitope, anuclear localization signal (NLS), and green fluorescent protein (GFP)attached to the C-terminus of Cas9 (FIG. 29A). Analysis of cellstransfected with a vector encoding the GFP-fused Cas9 revealed abundantCas9 expression and nuclear localization (FIG. 29B). Western blottingconfirmed that the Cas9 protein is expressed largely intact in extractsfrom these cells (FIG. 29A). To program Cas9, we expressed sgRNA bearinga 5′-terminal 20-nucleotide sequence complementary to the target DNAsequence, and a 42-nucleotide 3′-terminal stem loop structure requiredfor Cas9 binding (FIG. 29C). This 3′-terminal sequence corresponds tothe minimal stem-loop structure that has previously been used to programCas9 in vitro [8]. The expression of this sgRNA was driven by the humanU6 (RNA polymerase III) promoter [111]. Northern blotting analysis ofRNA extracted from cells transfected with the U6 promoter-driven sgRNAplasmid expression vector showed that the sgRNA is indeed expressed, andthat their stability is enhanced by the presence of Cas9 (FIG. 29D).

FIG. 29A-29E demonstrates that co-expression of Cas9 and guide RNA inhuman cells generates double-strand DNA breaks at the target locus.(FIG. 29A) Top; schematic diagram of the Cas9-HA-NLS-GFP expressionconstruct. Bottom; lysate from HEK293T cells transfected with the Cas9expression plasmid was analyzed by Western blotting using an anti-HAantibody. (FIG. 29B) Fluorescence microscopy of HEK293T cells expressingCas9-HA-NLS-GFP. (FIG. 29C) Design of a single-guide RNA (sgRNA, i.e., asingle-molecule DNA-targeting RNA) targeting the human CLTA locus. Top;schematic diagram of the sgRNA target site in exon 7 of the human CLTAgene. The target sequence that hybridizes to the guide segment of CLTA1sgRNA is indicated by “CLTA1 sgRNA.” The GG di-nucleotide protospaceradjacent motif (PAM) is marked by an arrow. Black lines denote the DNAbinding regions of the control ZFN protein. The translation stop codonof the CLTA open reading frame is marked with a dotted line forreference. Middle; schematic diagram of the sgRNA expression construct.The RNA is expressed under the control of the U6 Pol III promoter and apoly(T) tract that serves as a Pol III transcriptional terminatorsignal. Bottom; sgRNA-guided cleavage of target DNA by Cas9. The sgRNAconsists of a 20-nt 5′-terminal guide segment followed by a 42-ntstem-loop structure required for Cas9 binding. Cas9-mediated cleavage ofthe two target DNA strands occurs upon unwinding of the target DNA andformation of a duplex between the guide segment of the sgRNA and thetarget DNA. This is dependent on the presence of a PAM motif(appropriate for the Cas9 being used, e.g., GG dinucleotide, see Example1 above) downstream of the target sequence in the target DNA. Note thatthe target sequence is inverted relative to the upper diagram. (FIG.29D) Northern blot analysis of sgRNA expression in HEK239T cells. (FIG.29E) Surveyor nuclease assay of genomic DNA isolated from HEK293T cellsexpressing Cas9 and/or CLTA sgRNA. A ZFN construct previously used totarget the CLTA locus [10] was used as a positive control for detectingDSB-induced DNA repair by non-homologous end joining.

Next we investigated whether site-specific DSBs are generated in HEK293Tcells transfected with Cas9-HA-NLS-mCherry and the CLTA1 sgRNA. To dothis, we probed for minor insertions and deletions in the locusresulting from imperfect repair by DSB-induced NHEJ using the Surveyornuclease assay [12]. The region of genomic DNA targeted by Cas9:sgRNA isamplified by PCR and the resulting products are denatured andreannealed. The rehybridized PCR products are incubated with themismatch recognition endonuclease Cel-1 and resolved on an acrylamidegel to identify Cel-1 cleavage bands. As DNA repair by NHEJ is typicallyinduced by a DSB, a positive signal in the Surveyor assay indicates thatgenomic DNA cleavage has occurred. Using this assay, we detectedcleavage of the CLTA locus at a position targeted by the CLTA1 sgRNA(FIG. 29E). A pair of ZFNs that target a neighboring site in the CLTAlocus provided a positive control in these experiments [10].

To determine if either Cas9 or sgRNA expression is a limiting factor inthe observed genome editing reactions, lysates prepared from thetransfected cells were incubated with plasmid DNA harboring a fragmentof the CLTA gene targeted by the CLTA1 sgRNA. Plasmid DNA cleavage wasnot observed upon incubation with lysate prepared from cells transfectedwith the Cas9-HA-NLS-GFP expression vector alone, consistent with theSurveyor assay results. However, robust plasmid cleavage was detectedwhen the lysate was supplemented with in vitro transcribed CLTA1 sgRNA(FIG. 30A). Furthermore, lysate prepared from cells transfected withboth Cas9 and sgRNA expression vectors supported plasmid cleavage, whilelysates from cells transfected with the sgRNA-encoding vector alone didnot (FIG. 30A). These results suggest that a limiting factor for Cas9function in human cells could be assembly with the sgRNA. We tested thispossibility directly by analyzing plasmid cleavage in lysates from cellstransfected as before in the presence and absence of added exogenoussgRNA. Notably, when exogenous sgRNA was added to lysate from cellstransfected with both the Cas9 and sgRNA expression vectors, asubstantial increase in DNA cleavage activity was observed (FIG. 30B).This result indicates that the limiting factor for Cas9 function inHEK293T cells is the expression of the sgRNA or its loading into Cas9.

FIG. 30A-30B demonstrates that cell lysates contain active Cas9:sgRNAand support site-specific DNA cleavage. (FIG. 30A) Lysates from cellstransfected with the plasmid(s) indicated at left were incubated withplasmid DNA containing a PAM and the target sequence complementary tothe CLTA1 sgRNA; where indicated, the reaction was supplemented with 10pmol of in vitro transcribed CLTA1 sgRNA; secondary cleavage with XhoIgenerated fragments of ˜2230 and ˜3100 bp fragments indicative ofCas9-mediated cleavage. A control reaction using lysate from cellstransfected with a ZFN expression construct shows fragments of slightlydifferent size reflecting the offset of the ZFN target site relative tothe CLTA1 target site. (FIG. 30B) Lysates from cells transfected withCas9-GFP expression plasmid and, where indicated, the CLTA1 sgRNAexpression plasmid, were incubated with target plasmid DNA as in FIG.30A in the absence or presence of in vitro-transcribed CLTA1 sgRNA.

As a means of enhancing the Cas9:sgRNA assembly in living cells, we nexttested the effect of extending the presumed Cas9-binding region of theguide RNA. Two new versions of the CLTA1 sgRNA were designed to includean additional six or twelve base pairs in the helix that mimics thebase-pairing interactions between the crRNA and tracrRNA (FIG. 31A).Additionally, the 3′-end of the guide RNA was extended by fivenucleotides based on the native sequence of the S. pyogenes tracrRNA[9]. Vectors encoding these 3′ extended sgRNAs under the control ofeither the U6 or H1 Pol III promoters were transfected into cells alongwith the Cas9-HA-NLS-GFP expression vector and site-specific genomecleavage was tested using the Surveyor assay (FIG. 31B). The resultsconfirmed that cleavage required both Cas9 and the CLTA1 sgRNA, but didnot occur when either Cas9 or the sgRNA were expressed alone.Furthermore, we observed substantially increased frequencies of NHEJ, asdetected by Cel-1 nuclease cleavage, while the frequency of NHEJmutagenesis obtained with the control ZFN pair was largely unchanged.

FIG. 31A-31B demonstrates that 3′ extension of sgRNA constructs enhancessite-specific NHEJ-mediated mutagenesis. (FIG. 31A) The construct forCLTA1 sgRNA expression (top) was designed to generate transcriptscontaining the original Cas9-binding sequence (v1.0), or dsRNA duplexesextended by 4 base pairs (v2.1) or 10 base pairs (v2.2). (FIG. 31B)Surveyor nuclease assay of genomic DNA isolated from HEK293T cellsexpressing Cas9 and/or CLTA sgRNA v1.0, v2.1 or v2.2. A ZFN constructpreviously used to target the CLTA locus [10] was used as a positivecontrol for detecting DSB-induced DNA repair by non-homologous endjoining.

The results thus provide the framework for implementing Cas9 as a facilemolecular tool for diverse genome editing applications. A powerfulfeature of this system is the potential to program Cas9 with multiplesgRNAs in the same cell, either to increase the efficiency of targetingat a single locus, or as a means of targeting several locisimultaneously. Such strategies would find broad application ingenome-wide experiments and large-scale research efforts such as thedevelopment of multigenic disease models.

Example 3: The tracrRNA and Cas9 Families of Type II CRISPR-Cas ImmunitySystems

We searched for all putative type II CRISPR-Cas loci currently existingin publicly available bacterial genomes by screening for sequenceshomologous to Cas9, the hallmark protein of the type II system. Weconstructed a phylogenetic tree from a multiple sequence alignment ofthe identified Cas9 orthologues. The CRISPR repeat length and geneorganization of cas operons of the associated type II systems wereanalyzed in the different Cas9 subclusters. A subclassification of typeII loci was proposed and further divided into subgroups based on theselection of 75 representative Cas9 orthologues. We then predictedtracrRNA sequences mainly by retrieving CRISPR repeat sequences andscreening for anti-repeats within or in the vicinity of the cas genesand CRISPR arrays of selected type II loci. Comparative analysis ofsequences and predicted structures of chosen tracrRNA orthologues wasperformed. Finally, we determined the expression and processing profilesof tracrRNAs and crRNAs from five bacterial species.

Materials and Methods

Bacterial Strains and Culture Conditions

The following media were used to grow bacteria on plates: TSA(trypticase soy agar, Trypticase™ Soy Agar (TSA II) BD BBL, BectonDickinson) supplemented with 3% sheep blood for S. mutans (UA159), andBHI (brain heart infusion, BD Bacto™ Brain Heart Infusion, BectonDickinson) agar for L. innocua (Clip11262). When cultivated in liquidcultures, THY medium (Todd Hewitt Broth (THB, Bacto, Becton Dickinson)supplemented with 0.2% yeast extract (Servabacter®) was used for S.mutans, BHI broth for L. innocua, BHI liquid medium containing 1%vitamin-mix VX (Difco, Becton Dickinson) for N. meningitidis (A Z2491),MH (Mueller Hinton Broth, Oxoid) Broth including 1% vitamin-mix VX forC. jejuni (NCTC 11168; ATCC 700819) and TSB (Tryptic Soy Broth, BD BBL™Trypticase™ Soy Broth) for F. novicida (U112). S. mutans was incubatedat 37° C., 5% CO₂ without shaking. Strains of L. innocua, N.meningitidis and F. novicida were grown aerobically at 37° C. withshaking. C. jejuni was grown at 37° C. in microaerophilic conditionsusing campygen (Oxoid) atmosphere. Bacterial cell growth was followed bymeasuring the optical density of cultures at 620 nm (OD₆₂₀ nm) atregular time intervals using a microplate reader (BioTek PowerWave™)

Sequencing of Bacterial Small RNA Libraries.

C. jejuni NCTC 11168 (ATCC 700819), F. novicida U112, L. innocuaClip11262, N. meningitidis A Z2491 and S. mutans UA159 were cultivateduntil mid-logarithmic growth phase and total RNA was extracted withTRIzol (Sigma-Aldrich). 10 μg of total RNA from each strain were treatedwith TURBO™ DNase (Ambion) to remove any residual genomic DNA. RibosomalRNAs were removed by using the Ribo-Zero™ rRNA Removal Kits@ forGram-positive or Gram-negative bacteria (Epicentre) according to themanufacturer's instructions. Following purification with the RNA Clean &Concentrator™-5 kit (Zymo Research), the libraries were prepared usingScriptMiner™ Small RNA-Seq Library Preparation Kit (Multiplex, Illumina®compatible) following the manufacturer's instructions. RNAs were treatedwith the Tobacco Acid Pyrophosphatase (TAP) (Epicentre). Columns fromRNA Clean & Concentrator™-5 (Zymo Research) were used for subsequent RNApurification and the Phusion® High-Fidelity DNA Polymerase (New EnglandBiolabs) was used for PCR amplification. Specific userdefined barcodeswere added to each library (RNA-Seq Barcode Primers(Illumina®-compatible) Epicentre) and the samples were sequenced at theNext Generation Sequencing (CSF NGS Unit; on the web at “csf.” followedby “ac.at”) facilty of the Vienna Biocenter, Vienna, Austria (Illuminasingle end sequencing).

Analysis of tracrRNA and crRNA Sequencing Data

The RNA sequencing reads were split up using the illumina2bam tool andtrimmed by (i) removal of Illumina adapter sequences (cutadapt 1.0) and(ii) removal of 15 nt at the 3′ end to improve the quality of reads.After removal of reads shorter than 15 nt, the cDNA reads were alignedto their respective genome using Bowtie by allowing 2 mismatches: C.jejuni (GenBank: NC_002163), F. novicida (GenBank: NC_008601), N.meningitidis (GenBank: NC_003116), L. innocua (GenBank: NC_003212) andS. mutans (GenBank: NC_004350). Coverage of the reads was calculated ateach nucleotide position separately for both DNA strands usingBEDTools-Version-2.15.0. A normalized wiggle file containing coverage inread per million (rpm) was created and visualized using the IntegrativeGenomics Viewer (IGV) tool (“www.” followed by“broadinstitute.org/igv/”) (FIG. 36A-36F). Using SAMTools flagstat⁸⁰ theproportion of mapped reads was calculated on a total of mapped 9914184reads for C. jejuni, 48205 reads for F. novicida, 13110087 reads for N.meningitidis, 161865 reads L. innocua and 1542239 reads for S. mutans. Afile containing the number of reads starting (5′) and ending (3′) ateach single nucleotide position was created and visualized in IGV. Foreach tracrRNA orthologue and crRNA, the total number of reads retrievedwas calculated using SAMtools.

Cas9 Sequence Analysis, Multiple Sequence Alignment and Guide TreeConstruction

Position-Specific Iterated (PSI)-BLAST program was used to retrievehomologues of the Cas9 family in the NCBI non redundant database.Sequences shorter than 800 amino acids were discarded. The BLASTClustprogram set up with a length coverage cutoff of 0.8 and a score coveragethreshold (bit score divided by alignment length) of 0.8 was used tocluster the remaining sequences (FIG. 38A-38B). This procedure produced78 clusters (48 of those were represented by one sequence only). One (orrarely a few representatives) were selected from each cluster andmultiple alignment for these sequences was constructed using the MUSCLEprogram with default parameters, followed by a manual correction on thebasis of local alignments obtained using PSI-BLAST and HHpred programs.A few more sequences were unalignable and also excluded from the finalalignments. The confidently aligned blocks with 272 informativepositions were used for maximum likelihood tree reconstruction using theFastTree program with the default parameters: JTT evolutionary model,discrete gamma model with 20 rate categories. The same program was usedto calculate the bootstrap values.

FIG. 38A-38B depict sequences that were grouped according to theBLASTclust clustering program. Only sequences longer than 800 aminoacids were selected for the BLASTclust analysis (see Materials andMethods). Representative strains harboring cas9 orthologue genes wereused. Some sequences did not cluster, but were verified as Cas9sequences due to the presence of conserved motifs and/or other cas genesin their immediate vicinity.

Analysis of CRISPR-Cas Loci

The CRISPR repeat sequences were retrieved from the CRISPRdb database orpredicted using the CRISPRFinder tool (Grissa I et al., BMCBioinformatics 2007; 8:172; Grissa I et al., Nucleic Acids Res 2007).The cas genes were identified using the BLASTp algorithm and/or verifiedwith the KEGG database (on the web at “www.” followed by kegg.jp/).

In Silico Prediction and Analysis of tracrRNA Orthologues

The putative antirepeats were identified using the Vector NTI@ software(Invitrogen) by screening for additional, degenerated repeat sequencesthat did not belong to the repeat-spacer array on both strands of therespective genomes allowing up to 15 mismatches. The transcriptionalpromoters and rho-independent terminators were predicted using the BDGPNeural Network Promoter Prediction program (“www.” followed byfruitfly.org/seq_tools/promoter.html) and the TransTermHP software,respectively. The multiple sequence alignments were performed using theMUSCLE program with default parameters. The alignments were analyzed forthe presence of conserved structure motifs using the RNAalifoldalgorithm of the Vienna RNA package 2.0.

Results

Type II CRISPR-Cas Systems are Widespread in Bacteria

In addition to the tracrRNA-encoding DNA and the repeat-spacer array,type II CRISPR-Cas loci are typically composed of three to four casgenes organized in an operon (FIG. 32A-32B). Cas9 is the signatureprotein characteristic for type II and is involved in the steps ofexpression and interference. Cas1 and Cas2 are core proteins that areshared by all CRISPR-Cas systems and are implicated in spaceracquisition. Csn2 and Cas4 are present in only a subset of type IIsystems and were suggested to play a role in adaptation. To retrieve amaximum number of type II CRISPR-Cas loci, containing tracrRNA, we firstscreened publicly available genomes for sequences homologous to alreadyannotated Cas9 proteins. 235 Cas9 orthologues were identified in 203bacterial species. A set of 75 diverse sequences representative of allretrieved Cas9 orthologues were selected for further analysis (FIG.32A-32B, FIG. 38A-38B, and Materials and Methods).

FIG. 32A-32B depict (FIG. 32A) a phylogenetic tree of representativeCas9 sequences from various organisms as well as (FIG. 32B)representative Cas9 locus architecture. Bootstrap values calculated foreach node are indicated. Same color branches represent selectedsubclusters of similar Cas9 orthologues. CRISPR repeat length innucleotides, average Cas9 protein size in amino acids (aa) and consensuslocus architecture are shown for every subcluster.*-gi|116628213**-gi|116627542†-gi|34557790‡-gi|34557932. Type II-A ischaracterized by cas9-csx12, cas1, cas2, cas4. Type II-B ischaracterized by cas9, cas1, cas2 followed by a csn2 variant. Type II-Cis characterized by a conserved cas9, cas1, cas2 operon (See also FIG.38A-38B).

Next, we performed a multiple sequence alignment of the selected Cas9orthologues. The comparative analysis revealed high diversities in aminoacid composition and protein size. The Cas9 orthologues share only a fewidentical amino acids and all retrieved sequences have the same domainarchitecture with a central HNH endonuclease domain and splittedRuvC/RNaseH domain. The lengths of Cas9 proteins range from 984(Campylobacter jejuni) to 1629 (Francisella novicida) amino acids withtypical sizes of ˜1100 or ˜1400 amino acids. Due to the high diversityof Cas9 sequences, especially in the length of the inter-domain regions,we selected only well-aligned, informative positions of the preparedalignment to reconstruct a phylogenetic tree of the analyzed sequences(FIG. 32A-32B and Materials and Methods). Cas9 orthologues grouped intothree major, monophyletic clusters with some outlier sequences. Theobserved topology of the Cas9 tree is well in agreement with the currentclassification of type II loci, with previously defined type II-A andtype II-B forming separate, monophyletic clusters. To furthercharacterize the clusters, we examined in detail the cas operoncompositions and CRISPR repeat sequences of all listed strains.

Cas9 Subclustering Reflects Diversity in Type II CRISPR-Cas LociArchitecture

A deeper analysis of selected type II loci revealed that the clusteringof Cas9 orthologue sequences correlates with the diversity in CRISPRrepeat length. For most of the type II CRISPR-Cas systems, the repeatlength is 36 nucleotides (nt) with some variations for two of the Cas9tree subclusters. In the type II-A cluster (FIG. 32A-32B) that comprisesloci encoding the long Cas9 orthologue, previously named Csx12, theCRISPR repeats are 37 nt long. The small subcluster composed ofsequences from bacteria belonging to the Bacteroidetes phylum (FIG.32A-32B) is characterized by unusually long CRISPR repeats, up to 48 ntin size. Furthermore, we noticed that the subclustering of Cas9sequences correlates with distinct cas operon architectures, as depictedin FIG. 32A-32B. The third major cluster (FIG. 32A-32B) and the outlierloci (FIG. 32A-32B), consist mainly of the minimum operon composed ofthe cas9, cas1 and cas2 genes, with an exception of some incomplete locithat are discussed later. All other loci of the two first major clustersare associated with a fourth gene, mainly cas4, specific to type II-A orcsn2-like, specific to type II-B (FIG. 32A-32B). We identified genesencoding shorter variants of the Csn2 protein, Csn2a, within locisimilar to type II-B S. pyogenes CRISPR01 and S. thermophilus CRISPR3(FIG. 32A-32B). The longer variant of Csn2, Csn2b, was found associatedwith loci similar to type II-B S. thermophilus CRISPR1 (FIG. 32A-32B).Interestingly, we identified additional putative cas genes encodingproteins with no obvious sequence similarity to previously describedCsn2 variants. One of those uncharacterized proteins is exclusivelyassociated with type II-B loci of Mycoplasma species (FIG. 32A-32B andFIG. 33A-33E). Two others were found encoded in type II-B loci ofStaphylococcus species (FIG. 33A-33E). In all cases the cas operonarchitecture diversity is thus consistent with the subclustering of Cas9sequences. These characteristics together with the general topology ofthe Cas9 tree divided into three major, distinct, monophyletic clusters,led us to propose a new, further division of the type II CRISPR-Cassystem into three subtypes. Type II-A is associated with Csx12-like Cas9and Cas4, type II-B is associated with Csn2-like and type II-C onlycontains the minimal set of the cas9, cas1 and cas2 genes, as depictedin FIG. 32A-32B.

FIG. 33A-33E depicts the architecture of type II CRISPR-Cas fromselected bacterial species. The vertical bars group the loci that codefor Cas9 orthologues belonging to the same tree subcluster (compare withFIG. 32A-32B). Horizontal black bar, leader sequence; black rectanglesand diamonds, repeat-spacer array. Predicted anti-repeats arerepresented by arrows indicating the direction of putative tracrRNAorthologue transcription. Note that for the loci that were not verifiedexperimentally, the CRISPR repeat-spacer array is considered here to betranscribed from the same strand as the cas operon. The transcriptiondirection of the putative tracrRNA orthologue is indicated accordingly.

In Silico Predictions of Novel tracrRNA Orthologues

Type II loci selected earlier based on the 75 representative Cas9orthologues were screened for the presence of putative tracrRNAorthologues. Our previous analysis performed on a restricted number oftracrRNA sequences revealed that neither the sequences of tracrRNAs northeir localization within the CRISPR-Cas loci seemed to be conserved.However, as mentioned above, tracrRNAs are also characterized by ananti-repeat sequence capable of base-pairing with each of the pre-crRNArepeats to form tracrRNA:precrRNA repeat duplexes that are cleaved byRNase III in the presence of Cas9. To predict novel tracrRNAs, we tookadvantage of this characteristic and used the following workflow: (i)screen for potential anti-repeats (sequence base-pairing with CRISPRrepeats) within the CRISPR-Cas loci, (ii) select anti-repeats located inthe intergenic regions, (iii) validate CRISPR anti-repeat:repeatbase-pairing, and (iv) predict promoters and Rho-independenttranscriptional terminators associated to the identified tracrRNAs.

To screen for putative anti-repeats, we retrieved repeat sequences fromthe CRISPRdb database or, when the information was not available, wepredicted the repeat sequences using the CRISPRfinder software. In ourprevious study, we showed experimentally that the transcriptiondirection of the repeat-spacer array compared to that of the cas operonvaried among loci. Here RNA sequencing analysis confirmed thisobservation. In some of the analyzed loci, namely in F. novicida, N.meningitidis and C. jejuni, the repeat-spacer array is transcribed inthe opposite direction of the cas operon (see paragraph ‘Deep RNAsequencing validates expression of novel tracrRNA orthologues’ and FIG.33A-33E and FIG. 34A-34B) while in S. pyogenes, S. mutans, S.thermophilus and L. innocua, the array and the cas operon aretranscribed in the same direction. These are the only type IIrepeat-spacer array expression data available to date. To predict thetranscription direction of other repeat-spacer arrays, we considered theprevious observation according to which the last repeats of the arraysare usually mutated. This remark is in agreement with the current spaceracquisition model, in which typically the first repeat of the array isduplicated upon insertion of a spacer sequence during the adaptationphase. For 37 repeat spacer arrays, we were able to identify the mutatedrepeat at the putative end of the arrays. We observed that the predictedorientation of transcription for the N. meningitidis and C. jejunirepeat-spacer array would be opposite to the orientation determinedexperimentally (RNA sequencing and Northern blot analysis). As thepredicted orientation is not consistent within the clusters and as inmost of the cases we could detect potential promoters on both ends ofthe arrays, we considered transcription of the repeat-spacer arrays tobe in the same direction as transcription of the cas operon, if notvalidated otherwise.

FIG. 34A-34B depicts tracrRNA and pre-crRNA co-processing in selectedtype II CRISPR Cas systems. CRISPR loci architectures with verifiedpositions and directions of tracrRNA and pre-crRNA transcription areshown. Top sequences, pre-crRNA repeats; bottom sequences, tracrRNAsequences base-pairing with crRNA repeats. Putative RNA processing sitesas revealed by RNA sequencing are indicated with arrowheads. For eachlocus, arrowhead sizes represent relative amounts of the retrieved 5′and 3′ ends (see also FIG. 37A-37O).

FIG. 37A-37O lists all tracrRNA orthologues and mature crRNAs retrievedby sequencing for the bacterial species studied, including coordinates(region of interest) and corresponding cDNA sequences (5′ to 3′). Thearrows represent the transcriptional direction (strand). Number of cDNAreads (calculated using SAMtools), coverage numbers (percentage ofmapped reads) and predominant ends associated with each transcript areindicated. Numbers of reads starting or stopping at each nucleotideposition around the 5′ and 3′ ends of each transcript are displayed. Thesizes of each crRNA mature forms are indicated. The number allocated toeach crRNA species corresponds to the spacer sequence position in thepre-crRNA, according to the CRISPRdb. The number allocated to eachtracrRNA species corresponds to different forms of the same transcript.

We then screened the selected CRISPR-Cas loci including sequenceslocated 1 kb upstream and downstream on both strands for possible repeatsequences that did not belong to the repeat-spacer array, allowing up to15 mismatches. On average, we found one to three degenerated repeatsequences per locus that would correspond to anti-repeats of tracrRNAorthologues and selected the sequences located within the intergenicregions. The putative anti-repeats were found in four typicallocalizations: upstream of the cas9 gene, in the region between cas9 andcas1, and upstream or downstream of the repeat-spacer array (FIG.33A-33E). For every retrieved sequence, we validated the extent ofbase-pairing formed between the repeat and anti-repeat (FIG. 44A-44C) bypredicting the possible RNA:RNA interaction and focusing especially oncandidates with longer and perfect complementarity region forming anoptimal double-stranded structure for RNase III processing. To predictpromoters and transcriptional terminators flanking the anti-repeat, weset the putative transcription start and termination sites to beincluded within a region located maximally 200 nt uptream and 100 ntdownstream of the anti-repeat sequence, respectively, based on ourprevious observations²⁶. As mentioned above, experimental information onthe transcriptional direction of most repeat-spacer arrays of type IIsystems is lacking. The in silico promoter prediction algorithms oftengive false positive results and point to putative promoters that wouldlead to the transcription of repeat-spacer arrays from both strands. Insome cases we could not predict transcriptional terminators, even thoughthe tracrRNA orthologue expression could be validated experimentally, asexemplified by the C. jejuni locus (see paragraph ‘Deep RNA sequencingvalidates expression of novel tracrRNA orthologues’). We suggest toconsider promoter and transcriptional terminator predictions only as asupportive, but not essential, step of the guideline described above.

FIG. 44A-44C depicts predicted pre-crRNA repeat:tracrRNA anti-repeatbasepairing in selected bacterial species. ^(b)The CRISPR loci belong tothe type II (Nmeni/CASS4) CRISPR-Cas system. Nomenclature is accordingto the CRISPR database (CRISPRdb). Note that S. thermophilus LMD-9 andW. succinogenes contain two type II loci. ^(c)Upper sequence, pre-crRNArepeat consensus sequence (5′ to 3′); lower sequence, tracrRNA homologuesequence annealing to the repeat (anti-repeat; 3′ to 5′). Note that therepeat sequence given is based on the assumption that the CRISPRrepeat-spacer array is transcribed from the same strand as the casoperon. For the sequences that were validated experimentally in thisstudy, RNA sequencing data were taken into account to determine thebase-pairing. See FIG. 33A-33E. ^(d)Two possible anti-repeats wereidentified in the F. tularensis subsp. novicida, W. succinogenes andgamma proteobacterium HTCC5015 type II-A loci. Upper sequence pairing,anti-repeat within the putative leader sequence; lower sequence pairing,anti-repeat downstream of the repeat spacer array. See FIG. 33A-33E. °Two possible anti-repeats were identified in the S. wadsworthensis typeII-A locus. Upper sequence pairing, anti-repeat; lower sequence pairing,anti-repeat within the putative leader sequence See FIG. 33A-33E.^(f)Two possible anti-repeats were identified in the L. gasseri typeII-B locus. Upper sequence pairing, anti-repeat upstream of cas9; lowersequence pairing, anti-repeat between the cas9 and cas1 genes. See FIG.33A-33E. ^(g)Two possible anti-repeats were identified in the C. jejunitype II-C loci. Upper sequence pairing, anti-repeat upstream of cas9;lower sequence pairing, anti-repeat downstream of the repeat-spacerarray. See FIG. 33A-33E. ^(h)Two possible anti-repeats were identifiedin the R. rubrum type II-C locus. Upper sequence pairing, antirepeatdownstream of the repeat-spacer array; lower sequence pairing,anti-repeat upstream of cas1. See FIG. 33A-33E.

A Plethora of tracrRNA Orthologues

We predicted putative tracrRNA orthologues for 56 of the 75 lociselected earlier. The results of predictions are depicted in FIG.33A-33E. As already mentioned, the direction of tracrRNA transcriptionindicated in this Figure is hypothetical and based on the indicateddirection of repeat-spacer array transcription. As previously stated,sequences encoding putative tracrRNA orthologues were identifiedupstream, within and downstream of the cas operon, as well as downstreamof the repeat spacer arrays, including the putative leader sequences,commonly found in type II-A loci (FIG. 33A-33E). However, we observedthat anti-repeats of similar localization within CRISPR-Cas loci can betranscribed in different directions (as observed when comparing e.g.Lactobacillus rhamnosus and Eubacterium rectale or Mycoplasma mobile andS. pyogenes or N. meningitidis) (FIG. 33A-33E). Notably, loci groupedwithin a same subcluster of the Cas9 guide tree share a commonarchitecture with respect to the position of the tracrRNA-encoding gene.We identified anti-repeats around the repeat-spacer array in type II-Aloci, and mostly upstream of the cas9 gene in types II-B and II-C withseveral notable exceptions for the putative tracrRNA located betweencas9 and cas1 in three distinct subclusters of type II-B.

Some Type II CRISPR-Cas Loci have Defective Repeat-Spacer Arrays and/ortracrRNA Orthologues

For six type II loci (Fusobacterium nucleatum, Aminomonas paucivorans,Helicobacter mustelae, Azospirillum sp., Prevotella ruminicola andAkkermansia muciniphila), we identified potential anti-repeats with weakbase-pairing to the repeat sequence or located within the open readingframes. Notably, in these loci, a weak anti-repeat within the openreading frame of the gene encoding a putative ATPase in A. paucivorans,a strong anti-repeat within the first 100 nt of the cas9 gene inAzospirillum sp. B510 and a strong anti-repeat overlapping with bothcas9 and cas1 in A. muciniphila were identified (FIG. 33A-33E). Fortwelve additional loci (Peptoniphilus duerdenii, Coprococcus catus,Acidaminococcus intestini, Catenibacterium mitsuokai, Staphylococcuspseudintermedius, Ilyobacter polytropus, Elusimicrobium minutum,Bacteroides fragilis, Acidothermus cellulolyticus, Corynebacteriumdiphteriae, Bifidobacterium longum and Bifidobacterium dentium), wecould not detect any putative anti-repeat. There is no availableinformation on pre-crRNA expression and processing in these CRISPR-Casloci. Thus, the functionality of type II systems in the absence of aclearly defined tracrRNA orthologue remains to be addressed. For sevenanalyzed loci we could not identify any repeat spacer array(Parasutterella excrementihominis, Bacillus cereus, Ruminococcus albus,Rhodopseudomonas palustris, Nitrobacter hamburgensis, Bradyrhizobium sp.and Prevotella micans) (FIG. 33A-33E) and in three of those(Bradyrhizobium sp. BTAi1, N. hamburgensis and B. cereus) we detectedcas9 as a single gene with no other cas genes in the vicinity. For thesethree loci, we failed to predict any small RNA sequence upstream ordownstream of the cas9 gene. In the case of R. albus and P.excrementihominis, the genomic contig containing cas9 is too short toallow prediction of the repeat spacer array.

Deep RNA Sequencing Validates Expression of Novel tracrRNA Orthologues

To verify the in silico tracrRNA predictions and determinetracrRNA:pre-crRNA coprocessing patterns, RNAs from selectedGram-positive (S. mutans and L. innocua) and Gram-negative (N.meningitidis, C. jejuni and F. novicida) bacteria were analyzed by deepsequencing. Sequences of tracrRNA orthologues and processed crRNAs wereretrieved (FIG. 36A-36F and FIG. 37A-37O). Consistent with previouslypublished differential tracrRNA sequencing data in S. pyogenes ²⁶,tracrRNA orthologues were highly represented in the libraries, rangingfrom 0.08 to 6.2% of total mapped reads. Processed tracrRNAs were alsomore abundant than primary transcripts, ranging from 66% to more than95% of the total amount of tracrRNA reads (FIG. 36A-36F and FIG.37A-37O).

FIG. 36A-36F depict the expression of bacterial tracrRNA orthologues andcrRNAs revealed by deep RNA sequencing. Expression profiles of tracrRNAorthologues and crRNAs of selected bacterial strains are representedalong the corresponding genomes by bar charts (Images captured from theIntegrative Genomics Viewer (IGV) tool). Campylobacter jejuni (GenBank:NC_002163), Francisella novicida (GenBank: NC_008601), Neisseriameningitidis (GenBank: NC_003116), Listeria innocua (GenBank: NC_003212)and Streptococcus mutans (GenBank: NC_004350). Genomic coordinates aregiven. ^(a)Sequence coverage calculated using BEDTools-Version-2.15.0(Scale given in reads per million). ^(b)Distribution of reads starting(5′) and ending (3′) at each nucleotide position are indicated (Scalegiven in numbers of reads). Upper panels correspond to transcripts fromthe positive strand and lower panels correspond to transcripts from thenegative strand. The negative coverage values and peaks presented belowthe axes indicate transcription from the negative strand of the genome.Predominant 5′- and 3′-ends of the reads are plotted for all RNAs. Notethat given the low quality of L. innocua cDNA library, the reads areshortened for crRNAs, and an accumulation of the reads at the 3′ end oftracrRNA is observed, presumably due to RNA degradation.

To assess the 5′ ends of tracrRNA primary transcripts, we analyzed theabundance of all 5′ end reads of tracrRNA and retrieved the mostprominent reads upstream or in the vicinity of the 5′ end of thepredicted anti-repeat sequence. The 5′ ends of tracrRNA orthologues werefurther confirmed using the promoter prediction algorithm. Theidentified 5′ ends of tracrRNAs from S. mutans, L. innocua and N.meningitidis correlated with both in silico predictions and Northernblot analysis of tracrRNA expression²⁶. The most prominent 5′ end of C.jejuni tracrRNA was identified in the middle of the anti-repeatsequence. Five nucleotides upstream, an additional putative 5′ endcorrelating with the in silico prediction and providing longer sequenceof interaction with the CRISPR repeat sequence was detected. Weretrieved relatively low amount of reads from the F. novicida librarythat corresponded almost exclusively to processed transcripts. Analysisof the very small amount of reads of primary transcripts provided a 5′end that corresponded to the strong in silico promoter predictions.Northern blot probing of F. novicida tracrRNA further confirmed thevalidity of the predictions showing the low abundance of transcripts ofaround 90 nt in length. The results are listed in Table 2. For allexamined species, except N. meningitidis, primary tracrRNA transcriptswere identified as single small Glean Version RNA species of 75 to 100nt in length. In the case of N. meningitidis, we found a predominantprimary tracrRNA form of ˜110 nt and a putative longer transcript of˜170 nt represented by a very low amount of reads and detectedpreviously as a weak band by Northern blot analysis.

TABLE 2 Selected tracrRNA orthologues 5′-end^(b) RNA-seq Most LengthStrains^(a) Transcript First read prominent Predicted 3′-end^(c) (nt) S.pyogenes primary — 854546 — 854376  ~171 SF370 primary — 854464 — ~89processed — 854450 — ~75 C. jejuni primary 1455497 1455502 14554971455570 ~75 NCTC 11168 processed — 1455509 — ~60 L. innocua primary2774774 2774774 2774773 2774863 ~90 Ciip11262 processed — 2774788 — ~75S. mutans primary 1335040 1335040 1355039 1335141  ~100 UA159 processed— 1335054 — ~85 1335062 ~80 N. meningitidia primary 614158 614162 614154614333  ~175 A Z2491 primary 614223 614225 614223  ~110 processed —614240 — ~90 F. novicida primary 817144 — 817145 817065 ~80 U112 817154processed — 817138 — ~75 817128 ~65 S. thermophilus primary — — 13843301384425 ~95 LMD-8 primary — — 646654 646762  ~110 P. multocida primary —— 1327287 1327396  ~110 Pm70 M. mobile primary — — 49470 49361  ~110163K ^(a)tracrRNA orthologues of S. thermophilus, P. multocida and M.mobile were predicted in silico. ^(b)RNA-seq, revealed by RNA sequencing(Table S3); first read, first 5′-end position retrieved by sequencing;most prominent, abundant 5′-end according to RNA-seq data; predicted, insilico prediction of transcription start site; underlined, 5′-end chosenfor the primary tracrRNA to be aligned. ^(c)Estimated 3′ end accordingto RNA-seq data and transcriptional terminator prediction.tracrRNA and Pre-crRNA Co-Processing Sites Lie in the Anti-Repeat:Repeat Region.

We examined the processed tracrRNA transcripts by analyzing abundanttracrRNA 5′ ends within the predicted anti-repeat sequence and abundantmature crRNA 3′ ends (FIG. 34A-34B and FIG. 45A-45B). In all species, weidentified the prominent 5′ ends of tracrRNA orthologues that couldresult from co-processing of the tracrRNA:pre-crRNA repeat duplexes byRNase III. We also identified the processed 5′-ends of crRNAs that mostprobably result from a second maturation event by putative trimming,consistently with previous observations. Noteworthy, in the closelyrelated RNA pairs of S. pyogenes, S. mutans and L. innocua, we observedthe same processing site around the G:C basepair in the middle of theanti-repeat sequence. In both S. mutans and L. innocua, we detectedadditional prominent tracrRNA 5′ ends and crRNA 3′ ends that couldsuggest further trimming of the tracrRNA:crRNA duplex, with 3′-end ofcrRNA being shortened additionally to the already mentioned 5′-endtrimming, following the RNase III-catalyzed first processing event.Similarly, in C. jejuni we found only a small amount of crRNA 3′ endsthat would fit to the RNase III processing patterns and retrieved thecorresponding 5′ ends of processed tracrRNA. Thus, the putative trimmingof tracrRNA:crRNA duplexes after initial cleavage by RNase III wouldresult in a shorter repeat-derived part in mature crRNAs, producingshorter tracrRNA:crRNA duplexes stabilized by a triple G:C base-pairingfor interaction with the endonuclease Cas9 and subsequent cleavage oftarget DNAs. The N. meningitidis RNA duplex seems to be processed at twoprimary sites further to the 3′ end of the CRISPR repeat, resulting in along repeat-derived part in mature crRNA and stable RNA:RNA interactiondespite the central bulge within the duplex. Interestingly, thetracrRNA:pre-crRNA duplex of F. novicida seems to be cleaved within theregion of low complementarity and some of the retrieved abundant 5′ endsof tracrRNA suggest its further trimming without concomitant trimming ofcrRNA. Differences in primary transcript sizes and in the location ofprocessing sites result in various lengths of processed tracrRNAsranging from −65 to 85 nt. The coordinates and sizes of the prominentprocessed tracrRNA transcripts are shown in Table 2 and FIG. 37A-37O.The observed processing patterns of tracrRNA and crRNA are well inagreement with the previously proposed model of two maturation events.The putative further trimming of some of the tracrRNA 5′-ends and crRNA3′-ends could stem from the second maturation event or alternatively, bean artifact of the cDNA library preparation or RNA sequencing. Thenature of these processings remains to be investigated further.

Sequences of tracrRNA Orthologues are Highly Diverse

Sequences similarities of selected tracrRNA orthologues were alsodetermined. We performed multiple sequence alignments of primarytracrRNA transcripts of S. pyogenes (89 nt form only), S. mutans, L.innocua and N. meningitidis (110 nt form only), S. thermophilus, P.multocida and M. mobile (Table 2, FIG. 35). We observed high diversityin tracrRNA sequences but significant conservation of sequences fromclosely related CRISPR-Cas loci. tracrRNAs from L. innocua, S. pyogenes,S. mutans and S. thermophiles share on average 77% identity andtracrRNAs from N. meningitidis and P. multocida share 82% identityaccording to pairwise alignments. The average identity of the analyzedtracrRNA sequences is 56%, comparable to the identity of random RNAsequences. This observation further confirms that the prediction oftracrRNA orthologues based on sequence similarity can be performed onlyin the case of closely related loci. We also sought for possibletracrRNA structure conservation but could not find any significantsimilarity except one co-variation and conserved transcriptionalterminator structure (FIG. 35).

FIG. 35 depicts sequence diversity of tracrRNA orthologues. tracrRNAsequence multiple alignment. S. thermophilus and S. thermophilus2,tracrRNA associated with SEQ ID NO:41 and SEQ ID NO:40 Cas9 orthologues,accordingly. Black, highly conserved; dark grey, conserved; light grey,weakly conserved. Predicted consensus structure is depicted on the topof the alignment. Arrows indicate the nucleotide covariations. S.pyogenes SF370, S. mutans UA159, L. innocua Clip11262, C. jejuni NCTC11168, F. novicida U112 and N. meningitidis A Z2491 tracrRNAs werevalidated by RNA sequencing and Northern blot analysis. S. thermophilesLMD-9 tracrRNA was validated by Northern blot analysis. P. multocidaPm70 tracrRNA was predicted from high similarity of the CRISPR-Cas locuswith that of N. meningitidis A Z2491. M. mobile 163K tracrRNA waspredicted in silico from strong predictions of transcriptional promoterand terminator.

Example 4: Repurposing CRISPR as an RNA-Guided Platform forSequence-Specific Control of Gene Expression

Targeted gene regulation on a genome-wide scale is a powerful strategyfor interrogating, perturbing and engineering cellular systems. Theinventors have developed a new method for controlling gene expression,based on Cas9, an RNA-guided DNA endonuclease from a Type II CRISPRsystem. This example demonstrates that a catalytically dead Cas9,lacking endonuclease activity, when co-expressed with a guide RNA,generates a DNA recognition complex that can specifically interfere withtranscriptional elongation, RNA polymerase binding, or transcriptionfactor binding. This system, called CRISPR interference (CRISPRi), canefficiently repress expression of targeted genes in Escherichia coliwith no detectable off-target effects. CRISPRi can be used to repressmultiple target genes simultaneously, and its effects are reversible. Inaddition, the system can be adapted for gene repression in mammaliancells. This RNA-guided DNA recognition platform provides a simpleapproach for selectively perturbing gene expression on a genome-widescale.

Materials and Methods

Strains and Media

The Escherichia coli K-12 strain MG1655 was used as the host strain forthe in vivo fluorescence measurements. An E. coli MG1655-derived strainthat endogenously expresses a variant of RNAP with a 3×-FLAG epitope tagattached to the C-terminal end of the RpoC subunit was used for allsequencing experiments. EZ rich defined media (EZ-RDM, Teknoka) was usedas the growth media for in vivo fluorescence assays. Genetictransformation and verification of transformation were done usingstandard protocols, using AmpR, CmR, or KanR genes as selectablemarkers.

Plasmid Construction and E. coli Genome Cloning

The Cas9 and dCas9 genes were cloned from the previous described vectorpMJ806 and pMJ841, respectively. The genes were PCR amplified andinserted into a vector containing an anhydrotetracycline (aTc)-induciblepromoter PLtetO-1, a chloramphenicol selectable marker and a p15Areplication origin. The sgRNA template was cloned into a vectorcontaining a minimal synthetic promoter (J23119) with an annotatedtranscription start site, an ampicillin selectable marker and a ColE1replication origin. Inverse PCR was used to generate sgRNA cassetteswith new 20-bp complementary regions. To insert fluorescent reportergenes into E. coli genomes, the fluorescence gene was first cloned ontoan entry vector, which was then PCR amplified to generate linearized DNAfragments that contained nsfA 5′/3′ UTR sequences, the fluorescent geneand a KanR selectable marker. The E. coli MG1655 strain was transformedwith a temperature-sensitive plasmid pKD46 that contained λ-Redrecombination proteins (Exo, Beta and Gama). Cell cultures were grown at30° C. to an OD (600 nm) of ˜0.5, and 0.2% arabinose was added to induceexpression of the λ-Red recombination proteins for 1 h. The cells wereharvested at 4° C., and used for transformation of the linearized DNAfragments by electroporation. Cells that contain correct genomeinsertions were selected by using 50 μg/mL Kanamycin.

Flow Cytometry and Analysis

Strains were cultivated in EZ-RDM containing 100 μg/mL carbenicillin and34 μg/mL chloramphenicol in 2 mL 96-well deep well plates (Costar 3960)overnight at 37 □C and 1200 r.p.m. One-μL of this overnight culture wasthen added to 249 μL of fresh EZ-RDM with the same antibioticconcentrations with 2 μM aTc supplemented to induce production of thedCas9 protein. When cells were grown to mid-log phase (˜4 h), the levelsof fluorescence protein were determined using the LSRII flow cytometer(BD Biosciences) equipped with a high-throughput sampler. Cells weresampled with a low flow rate until at least 20,000 cells had beencollected. Data were analyzed using FCS Express (De Novo Software) bygating on a polygonal region containing 60% cell population in theforward scatter-side scatter plot. For each experiment, triplicatecultures were measured, and their standard deviation was indicated asthe error bar.

B-Galactosidase Assay

To perform β-galactosidase assay, 1 μL of overnight culture prepared asabove was added to 249 μL of fresh EZ-RDM with the same antibioticconcentrations with 2 μM aTc, with or without 1 mM Isopropylβ-D-1-thiogalactopyranoside (IPTG). Cells were grown to mid-log phase.The LacZ activity of 100 uL of this culture was measured using the yeastβ-galactosidase assay kit (Pierce) following the instructions.

Extraction and Purification of Total RNA

For each sample, a monoclonal culture of E. coli was grown at 37° C.from an OD (600 nm) 0.1 in 500 mL of EZ-RDM to early log-phase (OD0.45±0.05), at which point the cells were harvested by filtration over0.22 μm nitrocellulose filters (GE) and frozen in liquid nitrogen tosimultaneously halt all transcriptional progress. Frozen cells (100 μg)were pulverized on a Qiagen TissueLyser II mixer mill 6 times at 15 Hzfor 3 min in the presence of 500 μL frozen lysis buffer (20 mM Tris pH8, 0.4% Triton X-100, 0.1% NP-40, 100 mM NH₄Cl, 50 U/mL SUPERase⋅In(Ambion) and 1× protease inhibitor cocktail (Complete, EDTA-free,Roche), supplemented with 10 mM MnCl₂ and 15 μM Tagetin transcriptionalinhibitor (Epicentre).

The lysate was resuspended on ice by pipetting. RQ1 DNase I (110 Utotal, Promega) was added and incubated for 20 min on ice. The reactionwas quenched with EDTA (25 mM final) and the lysate clarified at 4° C.by centrifugation at 20,000 g for 10 min. The lysate was loaded onto aPD MiniTrap G-25 column (GE Healthcare) and eluted with lysis buffersupplemented with 1 mM EDTA.

Total mRNA Purification

Total RNA was purified from the clarified lysate using the miRNeasy kit(Qiagen). 1 μg of RNA in 20 μL of 10 mM Tris pH 7 was mixed with anequal volume of 2× alkaline fragmentation solution (2 mM EDTA, 10 mMNa₂CO₃, 90 mM NaHCO₃, pH 9.3) and incubated for ˜25 min at 95° C. togenerate fragments ranging from 30-100 nt. The fragmentation reactionwas stopped by adding 0.56 mL of ice-cold precipitation solution (300 mMNaOAc pH 5.5 plus GlycoBlue (Ambion)), and the RNA was purified by astandard isopropanol precipitation. The fragmented mRNA was thendephosphorylated in a 50 μL reaction with 25 U T4 PNK (NEB) in 1×PNKbuffer (without ATP) plus 0.5 U SUPERase⋅In, and precipitated withGlycoBlue via standard isopropanol precipitation methods.

Nascent RNA Purification

For nascent RNA purification, the clarified lysate was added to 0.5 mLanti-FLAG M2 affinity gel (Sigma Aldrich) as described previously. Theaffinity gel was washed twice with lysis buffer supplemented with 1 mMEDTA before incubation with the clarified lysate at 4° C. for 2.5 h withnutation. The immunoprecipitation was washed 4×10 ml with lysis buffersupplemented with 300 mM KCl, and bound RNAP was eluted twice with lysisbuffer supplemented with 1 mM EDTA and 2 mg/mL 3×-FLAG peptide (SigmaAldrich). Nascent RNA was purified from the eluate using the miRNeasykit (Qiagen) and converted to DNA using a previously established librarygeneration protocol.

DNA Library Preparation and DNA Sequencing

The DNA library was sequencing on an Illumina HiSeq 2000. Reads wereprocessed using the HTSeq Python package and other custom softwarewritten in Python. The 3′-end of the sequenced transcript was aligned tothe reference genome using Bowtie (“bowtie-bio” preceeding“.sourceforge.net”) and the RNAP profiles generated in MochiView(“johnsonlab.ucsf” preceeding “.edu/mochi.html”).

Plasmid Design and Construction for CRISPRi in Human Cells

The sequence encoding mammalian codon optimized Streptococcus pyogenesCas9 (DNA 2.0) was fused with three C-terminal SV40 nuclear localizationsequences (NLS) or to tagBFP flanked by two NLS. Using standard ligationindependent cloning we cloned these two fusion proteins into MSCV-Puro(Clontech). Guide sgRNAs were expressed using a lentiviral U6 basedexpression vector derived from pSico which co-expresses mCherry from aCMV promoter. The sgRNA expression plasmids were cloned by insertingannealed primers into the lentiviral U6 based expression vector that wasdigested by BstXI and XhoI.

Cell Culture, DNA Transfections and Fluorescence Measurements forCRISPRi in Human Cells

HEK293 cells were maintained in Dulbecco's modified eagle medium (DMEM)in 10% FBS, 2 mM glutamine, 100 units/mL streptomycin and 100 μg/mLpenicillin. HEK293 were infected with a GFP expressing MSCV retrovirususing standard protocols and sorted by flow cytometry using a BD FACSAria2 for stable GFP expression. GFP expressing HEK293 cells weretransiently transfected using TransIT-LT1 transfection reagent (Mirus)with the manufacturers recommended protocol in 24 well plates using 0.5μg of the dCas9 expression plasmid and 0.5 μg of the RNA expressionplasmid (with 0.25 μg of GFP reporter plasmid for FIG. 45B). 72 hoursfollowing transfection, cells were trypsinized to a single cellsuspension. The U6 vector contains a constitutive CMV promoter driving amCherry gene. GFP expression was analyzed using a BD LSRII FACS machineby gating on the mCherry positive populations (>10-fold brighter mCherryover the negative control cells).

Designed RNAs

sgRNA designs used in the Figures: only the 20 nucleotide matchingregion (DNA targeting segment) are listed (unless otherwise noted):

The mRFP-targeting sgRNAs used in FIG. 40C (SEQ ID NOs:741-746);

The promoter-targeting sgRNAs used in FIG. 40D (SEQ ID NOs:747-751);

Target promoter sequence in FIG. 40D (SEQ ID NO:752);

The mRFP-targeting sgRNAs used in FIG. 43B (SEQ ID NOs:753-760);

The sfGFP-targeting sgRNA (gfp) used in FIG. 42B (SEQ ID NO:761);

The sfGFP-targeting sgRNAs used in FIG. 43B (SEQ ID NOs:762-769);

The double-sgRNA targeting experiments in FIG. 43F and FIG. 51A-51C (SEQID NOs:770-778);

The lac operon-targeting sgRNAs used in FIG. 44B (SEQ ID NOs:779-787);and

The EGFP-targeting sgRNAs used in FIG. 45A-45B (SEQ ID NOs:788-794).

TABLE 3 Sequences used in the FIGS. of Example 4 (listed above) SequenceSEQ ID NO: T1 741 T2 742 T3 743 NT1 744 NT2 745 NT3 746 P1 747 P2 748 P3749 P4 750 P5 751 R1 770 R2 771 R3 772 R4 773 R5 774 R6 775 R7 776 R8777 R9 778 lacZ 779 lacI 780 lacY 781 lacA 782 crp 783 cya 784 A site785 O site 786 P site 787 eT1 788 eT2 789 eNT1 790 eNT2 791 eNT3 792eNT4 793 eNT5 794Results

The CRISPR (clustered regularly interspaced short palindromic repeats)system provides a new potential platform for targeted gene regulation.About 40% of bacteria and 90% of archaea possessCRISPR/CRISPR-associated (Cas) systems to confer resistance to foreignDNA elements. CRISPR systems use small base-pairing RNAs to target andcleave foreign DNA elements in a sequence-specific manner. There arediverse CRISPR systems in different organisms, and one of the simplestis the type II CRISPR system from Streptococcus pyogenes: only a singlegene encoding the Cas9 protein and two RNAs, a mature CRISPR RNA (crRNA)and a partially complementary trans-acting RNA (tracrRNA), are necessaryand sufficient for RNA-guided silencing of foreign DNAs (FIG. 46).Maturation of crRNA requires tracrRNA and RNase III. However, thisrequirement can be bypassed by using an engineered small guide RNA(sgRNA) containing a designed hairpin that mimics the tracrRNA-crRNAcomplex. Base pairing between the sgRNA and target DNA causesdouble-strand breaks (DSBs) due to the endonuclease activity of Cas9.Binding specificity is determined by both sgRNA-DNA basepairing and ashort DNA motif (protospacer adjacent motif or PAM, sequence: NGG)juxtaposed to the DNA complementary region. Thus, the CRISPR system onlyrequires a minimal set of two molecules—the Cas9 protein and the sgRNA,and therefore holds the potential to be used as a host-independentgene-targeting platform. It has been demonstrated that the Cas9/CRISPRcan be harnessed for site-selective RNA-guided genome editing (FIG.39A).

FIG. 46 depicts the mechanism of the type II CRISPR system from S.pyogenes. The system consists of a set of CRISPR-associated (Cas)proteins and a CRISPR locus that contains an array of repeat spacersequences. All repeats are the same and all spacers are different andcomplementary to the target DNA sequences. When the cell is infected byforeign DNA elements, the CRISPR locus will transcribe into a longprecursor transcript, which will be cleaved into smaller fragments. Thecleavage is mediated by a transacting antisense RNA (tracrRNA) and thehost RNase III. After cleavage, one single protein, Cas9, recognizes andbinds to the cleaved form of the crRNA. Cas9 guides crRNA to DNA andscans the DNA molecule. The complex is stabilized by basepairing betweenthe crRNA and the DNA target. In this case, Cas9 causes double-strandedDNA breaks due to its nuclease activity. This usually removes cognateDNA molecules, and cells confer immunity to certain DNA populations.

FIG. 39A-39B depicts the design of the CRISPR interference (CRISPRi)system. (FIG. 39A) The minimal interference system consists of a singleprotein and a designed sgRNA chimera. The sgRNA chimera consists ofthree domains (boxed region): a 20-nucleotide (nt) complementary regionfor specific DNA binding, a 42-nt hairpin for Cas9 binding (Cas9handle), and a 40-nt transcription terminator derived from S. pyogenes.The wild-type Cas9 protein contains the nuclease activity. The dCas9protein is defective in nuclease activity. (FIG. 39B) The wild-type Cas9protein binds to the sgRNA and forms a protein-RNA complex. The complexbinds to specific DNA targets by Watson-Crick basepairing between thesgRNA and the DNA target. In the case of wild-type Cas9, the DNA will becleaved due to the nuclease activity of the Cas9 protein. In the case ofnuclease defective Cas9, the complex disrupts appropriate transcription.A minimal CRISPRi system consists of a single protein and RNA and caneffectively silence transcription initiation and elongation

To implement such a CRISPRi platform in E. coli, the wild-type S.pyogenes Cas9 gene and an sgRNA were expressed from bacterial vectors todetermine if the system could perturb gene expression at a targetedlocus (FIG. 40A). The S. pyogenes CRISPR system is orthogonal to thenative E. coli system. The Cas9 protein is expressed from ananhydrotetracycline (aTc)-inducible promoter on a plasmid containing ap15A replication origin, and the sgRNA is expressed from a minimalconstitutive promoter on a plasmid containing a ColE1 replicationorigin. As an alternative strategy, a catalytically dead Cas9 mutant(dCas9), which is defective in DNA cleavage, was used and showed thatthis form of Cas9 still acts as a simple RNA-guided DNA binding complex.

FIG. 40A-40E demonstrates that CRISPRi effectively silencestranscription elongation and initiation. (FIG. 40A) The CRISPRi systemconsists of an inducible Cas9 protein and a designed sgRNA chimera. ThedCas9 contains mutations of the RuvC1 and HNH nuclease domains. ThesgRNA chimera contains three functional domains as described in FIG.39A-39B. (FIG. 40B) Sequence of designed sgRNA (NT1) and the DNA target.NT1 targets the non-template DNA strand of mRFP coding region. Only theregion surrounding the base-pairing motif (20-nt) is shown. Base-pairingnucleotides are numbered and the dCas9-binding hairpin is overlined. ThePAM sequence is underlined. (FIG. 40C) CRISPRi blocked transcriptionelongation in a strand-specific manner. A synthetic fluorescence-basedreporter system containing an mRFP-coding gene was inserted into the E.coli MG1655 genome (the nsfA locus). Six sgRNAs that bind to either thetemplate DNA strand or the non-template DNA strand were co-expressedwith the dCas9 protein, with their effects on the target mRFP measuredby in vivo fluorescence assay. Only sgRNAs that bind to the non-templateDNA strand showed silencing (10⁻³⁰⁰-fold). The control showsfluorescence of the cells with dCas9 protein but without the sgRNA.(FIG. 40D) CRISPRi blocked transcription initiation. Five sgRNAs weredesigned to bind to different regions around an E. coli promoter(J23119). The transcription start site was labeled as +1. The dottedoval shows the initial RNAP complex that covers a 75-bp region from −55to +20. Only sgRNAs targeting regions inside the initial RNAP complexshowed repression (P1-P4). Unlike transcription elongation block,silencing was independent of the targeted DNA strand. (FIG. 40E) CRISPRiregulation was reversible. Both dCas9 and sgRNA (NT1) were under thecontrol of an aTc-inducible promoter. Cell culture was maintained duringexponential phase. At time T=0, 1 M of aTc was supplemented to cellswith OD=0.001. Repression of target mRFP started within 10 min. Thefluorescence signal decayed in a way that is consistent with cellgrowth, suggesting the decay was due to cell division. In 240 min, thefluorescence reached the fully repressed level. At T=370 min, aTc iswashed away from the growth media, and cells were diluted back toOD=0.001. Fluorescence started to increase after 50 min, and took about300 min to rise to the same level as the positive control. Positivecontrol: always without the inducer; negative control: always with 1 μMaTc inducer. Fluorescence results in 2C, 2D, and 2E represent averageand SEM of at least three biological replicates. See also FIG. 47A-47Band FIG. 48.

The sgRNA molecules co-expressed with Cas9 each consist of threesegments: a 20-nucleotide (nt) target-specific complementary region, a42-nt Cas9 binding hairpin (Cas9 handle) and a 40-nt transcriptionterminator derived from S. pyogenes (FIG. 40B). A red fluorescentprotein (mRFP)-based reporter system, was inserted it into the E. coliMG1655 genome.

Co-expression of the wild-type Cas9 protein and an sgRNA (NT1) targetedto the mRFP coding sequence dramatically decreased transformationefficiency, likely due to Cas9-induced double-stranded breaks on thegenome (FIG. 47A). Sequencing of a few survivor colonies showed thatthey all had sequence rearrangements around the target mRFP site on thegenome, suggesting that there was strong selection against expression ofwild-type Cas9 and an sgRNA targeted to a host sequence. The dCas9mutant gene (non-cleaving), which contained two silencing mutations ofthe RuvC1 and HNH nuclease domains (D10A and H841A), alleviated thislethality, as verified by transformation efficiency and E. coli growthrates (FIG. 47A & FIG. 47B).

FIG. 47A-47B is related to FIG. 40A-40E and shows Growth curves of E.coli cell cultures co-transformed with dCas9 and sgRNA. (FIG. 47A)Transformation efficiency for transforming E. coli cells with twoplasmids. One plasmid contains an sgRNA that targets to a genomic copyof mRFP and the other plasmid contains the wild-type Cas9 or dCas9.Co-transformation of wild-type Cas9 and sgRNA is highly toxic, which canbe alleviated using dCas9. (FIG. 47B) The sgRNA (NT1) is designed totarget the coding sequence of mRFP. Co-expression of dCas9 and sgRNAexhibits almost no effects on cellular growth rates, suggesting thedCas9-sgRNA interaction with DNA is strong enough to block RNApolymerase but not DNA polymerase or cell replication. The resultsrepresent average and SEM of at least three independent experiments.

To test whether the dCas9:sgRNA complex could yield highly efficientrepression of gene expression, sgRNAs complementary to different regionsof the mRFP coding sequence were designed, either binding to thetemplate DNA strand or the non-template DNA strand. The resultsindicated that sgRNAs targeting the non-template DNA strand demonstratedeffective gene silencing (10 to 300-fold of repression), while thosetargeting the template strand showed little effect (FIG. 40C). Thesystem exhibited similar repression effects for genes that were withinthe E. coli genome or on a high-copy plasmid (FIG. 48). Furthermore,targeting to the promoter region also led to effective gene silencing(FIG. 40D). Targeting of the sgRNA to the −35 box significantly knockeddown gene expression (P1, ˜100-fold of repression), whereas targeting toother adjacent regions showed a dampened effect (P2-P4). Targetingsequences about 100-bp upstream of the promoter showed no effects (P5).Unlike targeting the coding sequence, when targeting the promoter, theefficiency of silencing is independent of the DNA strand; targeting oftemplate or non-template strands is equally effective (P2 and P3).

FIG. 48 is related to FIG. 40C and shows that CRISPRi could silenceexpression of a reporter gene on a multiple-copy plasmid. The mRFP genewas cloned onto a p15A plasmid. Presence of the dCas9 and anmRFP-specific sgRNA (NT1) strongly represses mRFP (˜300-fold). Therepression effect is similar to that observed using the mRFP in thegenome (FIG. 40C). Silencing is only effective when the sgRNA acts onthe nontemplate DNA strand but not the template DNA strand (T1). Also,silencing is highly specific, as a GFP-specific 3 sgRNA (gfp) shows noeffect on mRFP expression. Fluorescence results represent average andSEM of at least three biological replicates.

CRISPRi Gene Knockdown is Inducible and Reversible

Unlike gene knockout methods, one advantage of using CRISPRi-based knockdown of gene expression is the fact that this perturbation should bereversible. To test if CRISPRi regulation could be induced andsubsequently reversed, both dCas9 and mRFP-specific sgRNA (NT1) wereplaced under the control of the aTc-inducible promoter, and time-coursemeasurements of CRISPRi-mediated regulation of mRFP in response toinducers were performed (FIG. 40E). At time zero, cell culture that grewto the early exponential phase without inducers was supplemented with 1μM of aTc. The data indicated that the system could quickly respond tothe presence of inducers—the fluorescent reporter protein signal startedto decrease within 10 min of the addition of the inducer molecule.Because the mRFP protein is stable, the rate of fluorescence signaldecrease is limited by protein dilution due to cell growth, as seen by asimilar cell doubling time and loss of fluorescence half-time (both ˜36min). At 240 min, all cells were uniformly repressed to the same levelas the negative control. At 420 min, the inducer was washed away fromthe growth media and cells were diluted back to a lower OD. After adelay of 50 min, mRFP fluorescence started to increase. It took a total300 min for single-cell fluorescence to increase to the same level asthe positive control. The 50 min delay is most likely determined by thedCas9/sgRNA turnover rate offset by dilution by cell growth anddivision. In summary, these results demonstrate that the silencingeffects of dCas9-sgRNA can be induced and reversed.

Native Elongating Transcript Sequencing (NET-Seq) Confirms that CRISPRiFunctions by Blocking Transcription

dCas9 appeared to be functioning as an RNA-guided DNA binding complexthat could block RNA polymerase (RNAP) binding during transcriptionelongation. Since the non-template DNA strand shares the same sequenceidentity as the transcribed mRNA and only sgRNAs that bind to thenon-template DNA strand exhibited silencing, it remained a possibilitythat the dCas9:sgRNA complex interacts with mRNA and alters itstranslation or stability. To distinguish these possibilities, a recentlydescribed native elongating transcript sequencing (NET-seq) approach wasapplied to E. coli, which could be used to globally profile thepositions of elongating RNA polymerases and monitor the effect of thedCas9:sgRNA complex on transcription. In this NET-seq method, theCRISPRi system was transformed into an E. coli MG1655-derived strainthat contained a FLAG-tagged RNAP. The CRISPRi contained an sgRNA (NT1)that binds to the mRFP coding region. In vitro immunopurification of thetagged RNAP followed by sequencing of the nascent transcripts associatedwith elongating RNAPs allowed for distinguishing the pause sites of theRNAP.

These experiments demonstrated that the sgRNA induced strongtranscriptional pausing upstream of the sgRNA target locus (FIG. 41A).The distance between the pause site and the target site is 19-bp, whichis in perfect accordance with the previously reported ˜18-bp distancebetween the nucleotide incorporation of RNAP and its front-edge. Thisfinding is consistent with a mechanism of CRISPRi in which thetranscription block is due to physical collision between the elongatingRNAP and the dCas9:sgRNA complex (FIG. 41B). Binding of the dCas9:sgRNAcomplex to the template strand had little repressive effect, suggestingthat RNAP was able to read through the complex in this particularorientation. In this case, the sgRNA faces the RNAP, which might beunzipped by the helicase activity of RNAP. These experiments havedemonstrated that CRISPRi utilizes RNAs to directly block transcription.This mechanism is distinct from that of RNAi, for which knockdown ofgene expression requires the destruction of already transcribedmessenger RNAs, prior to their translation.

FIG. 41A-41B demonstrates that CRISPRi functions by blockingtranscription elongation. (FIG. 41A) FLAG-tagged RNAP molecules wereimmunoprecipitated and the associated nascent mRNA transcripts weresequenced. The top panel shows sequencing results of the nascent mRFPtranscript in cells without sgRNA, and the bottom panel shows results incells with sgRNA. In the presence of sgRNA, a strong transcriptionalpause was observed 19-bp upstream of the target site, after which thenumber of sequencing reads drops precipitously. (FIG. 41B) A proposedCRISPRi mechanism based on physical collision between RNAP anddCas9-sgRNA. The distance from the center of RNAP to its front edge is˜19 bp, which matches well with our measured distance between thetranscription pause site and 3′ of sgRNA basepairing region. The pausedRNAP aborts transcription elongation upon encountering the dCas9-sgRNAroadblock.

CRISPRi sgRNA-Guided Gene Silencing is Highly Specific

To evaluate the specificity of CRISPRi on a genome-wide scale, wholetranscriptome shotgun sequencing (RNA-seq) of dCas9-transformed cellswith and without sgRNA co-expression was performed (FIG. 42A). In thepresence of the sgRNA targeted to mRFP (NT1), the mRFP transcript wasthe sole gene exhibiting a decrease in abundance. No other genes showedsignificant change in expression upon addition of the sgRNA, withinsequencing errors. We also performed RNA-seq on cells with differentsgRNAs that target different genes. None of these experiments showedsignificant changes of genes besides the targeted gene (FIG. 49A-49C).Thus sgRNA-guided gene targeting and regulation is highly specific anddoes not have significant off-target effects.

FIG. 42A-42C demonstrates the targeting specificity of the CRISPRisystem. (FIG. 42A) Genome-scale mRNA sequencing (RNA-seq) confirmed thatCRISPRi targeting has no off-target effects. The sgRNA NT1 that binds tothe mRFP coding region was used. The dCas9, mRFP, and sfGFP genes arehighlighted. (FIG. 42B) Multiple sgRNAs can independently silence twofluorescent protein reporters in the same cell. Each sgRNA specificallyrepressed its cognate gene but not the other gene. When both sgRNAs werepresent, both genes were silenced. Error bars represent SEM from atleast three biological replicates. (FIG. 42C) Microscopic images forusing two sgRNAs to control two fluorescent proteins. The top panelshows the bright-field images of the E. coli cells, the middle panelshows the RFP channel, and the bottom shows the GFP panel. Co-expressionof one sgRNA and dCas9 only silences the cognate fluorescent protein butnot the other. The knockdown effect was strong, as almost nofluorescence was observed from cells with certain fluorescent proteinsilenced. Scale bar, 10 m. Control shows cells without any fluorescentprotein reporters. Fluorescence results represent average and SEM of atleast three biological replicates. See also FIG. 49A-49C.

FIG. 49A-49C is related to FIG. 42A and depicts the RNA-seq data ofcells with sgRNAs that target different genes. (FIG. 49A) (+/−) sgRNAthat targets the promoter of the endogenous lacI gene in E. coli. Thesame lac-targeting sgRNA was used as in FIG. 44A. (FIG. 49B) (+/−) 1 mMIPTG for cells without auto-inhibited sgRNA (sgRNA repressed its ownpromoter). (FIG. 49C) (+/−) sgRNA that targets the endogenous lacZ genein E. coli. The same lacZ-targeting sgRNA was used as in FIG. 44A. 1 mMIPTG was also supplemented to cells with the lacZ-targeting sgRNA.

CRISPRi can be Used to Simultaneously Regulate Multiple Genes

The CRISPRi system can allow control of multiple genes independentlywithout crosstalk. A dual-color fluorescence-reporter system based onmRFP and sfGFP was devised. Two sgRNAs with distinct complementaryregions to each gene were designed. Expression of each sgRNA onlysilenced the cognate gene and had no effect on the other. Co-expressionof two sgRNAs knocked down both genes (FIGS. 42B & 42C). These resultssuggest that the sgRNA-guided targeting is specific, with thespecificity dictated by its sequence identity, and not impacted by thepresence of other sgRNAs. This behavior should enable multiplex controlof multiple genes simultaneously by CRISPRi.

Factors that Determine CRISPRi Silencing Efficiency

To find determinants of CRISPRi targeting efficiency, the role oflength, sequence complementarity and position on silencing efficiencywas investigated (FIG. 43A). As suggested in FIG. 40C, the location ofthe sgRNA target sequence along the gene was important for efficiency.sgRNAs were further designed to cover the full length of the codingregions for both mRFP and sfGFP (Supplemental Data for sgRNA sequences).In all cases, repression was inversely correlated with the targetdistance from the transcription start site (FIG. 43B). A strong linearcorrelation was observed for mRFP. A similar, but slightly weakercorrelation was observed when sfGFP was used as the target, perhapsindicating varying kinetics of the RNA polymerase during differentpoints in elongation of this gene.

The sgRNA contains a 20-bp region complementary to the target. Toidentify the importance of this basepairing region, the length of sgRNANT1 was altered (FIG. 43C). While extension of the region from the 5′end did not affect silencing, truncation of the region severelydecreased repression. The minimal length of the basepairing regionneeded for gene silencing was 12 bp, with further truncation leading tocomplete loss of function. Single mutations were introduced into thebasepairing region of sgRNA NT1 and the overall effect on silencing wastested. From the results, three sub-regions could be discerned, eachwith a distinct contribution to the overall binding and silencing (FIG.43D). Any single mutation of the first 7 nucleotides dramaticallydecreased repression, suggesting this sequence constitutes a “seedregion” for binding, as noted previously for both the type I and type IICRISPR systems. Adjacent nucleotides were also mutated in pairs (FIG.43E and FIG. 50A-50E). In most cases, the relative repression activitydue to a double mutation was multiplicative, relative to the effects ofthe single mutants, suggesting an independent relationship between themismatches. Furthermore, in agreement with previous results on theimportance of the PAM sequence, an incorrect PAM totally abolishedsilencing even with a 20-bp perfect binding region (FIG. 43E). Thus, thespecificity of the CRISPRi system is determined jointly by the PAM(2-bp) and at least a 12-bp sgRNA-DNA stretch, the space of which islarge enough to cover most bacterial genomes for unique target sites.

Two sgRNAs both targeting the same gene were tested (FIG. 43F and FIG.51A-51C). Depending on the relative positioning of multiple sgRNAs,distinct combinatorial effects were observed. Combining two sgRNAs, eachwith about 300-fold repression, allowed for increased overall silencingup to a thousand-fold. Combining two weaker sgRNAs (˜5-fold) showedmultiplicative effects when used together. Suppressive combinatorialeffects were observed when using two sgRNAs whose targets overlapped.This was probably due to competition of both sgRNAs for binding to thesame region.

FIG. 43A-43F depicts the characterization of factors that affectsilencing efficiency. (FIG. 43A) The silencing effects were measured ofsgRNAs with different targeting loci on the same gene (distance from thetranslation start codon) and sgRNAs with different lengths of thebasepairing region to the same target locus (based on NT1). (FIG. 43B)The silencing efficiency was inversely correlated with the targetdistance from the translation start codon (orange—mRFP & green—sfGFP).The relative repression activity was calculated by normalizingrepression of each sgRNA to that of the sgRNA with the highestrepression fold change. Error bars represent SEM from three biologicalreplicates. (FIG. 43C) The length of the Watson-Crick basepairing regionbetween the sgRNA and the target DNA affects repression efficiency.Extensions of the basepairing region all exhibited strong silencingeffect, and truncations dramatically decreased repression. The minimallength of the basepairing region for detectable repression is 12-bp.Error bars represent SEM from three biological replicates. (FIG. 43D)Single mismatches were introduced into every nucleotide on sgRNA (NT1,FIG. 40B) how these single mismatches affected repression efficiency wasmeasured. Three sub-regions with distinct importance to the overallsilencing can be discerned. They show a step function. The first7-nuleotide region was critical for silencing, and likely constitutes a“seed” region for probing sgRNAs binding to the DNA target. The PAMsequence (NGG) was indispensible for silencing. Error bars represent SEMfrom three biological replicates. (FIG. 43E) Silencing effects of sgRNAswith adjacent double mismatches. The relative repression activity ofsingle-mismatched sgRNAs is shown with the mismatch position labeled onthe bottom. Experimentally measured activity of double-mismatched sgRNAsis shown. Calculated activity by multiplying the effects of twosingle-mismatched sgRNAs is shown in white and labeled with “Com.” Inmost cases, the silencing activity of a double-mismatched sgRNA wassimply a multiplication of the activities of single-mismatched sgRNAs(except FIG. 50B), suggesting an independent relationship between singlemismatches. Error bars represent SEM from three biological replicates.(FIG. 43F) Combinatorial silencing effects of using double sgRNAs totarget a single mRFP gene. Using two sgRNAs that target the same gene,the overall knockdown effect can be improved to almost 1,000-fold. Whentwo sgRNAs bind to non-overlapping sequences of the same gene,repression was augmented. When two sgRNAs target overlapping regions,repression was suppressed. Error bars represent SEM from threebiological replicates.

FIG. 50A-50E is related to FIG. 43E and depicts the silencing effects ofsgRNAs with adjacent double mismatches. The relative repression activityof single-mismatched sgRNAs is shown with the mismatch position labeledon the bottom. Experimentally measured activity of double-mismatchedsgRNAs is also shown. Activity calculated by multiplying the effects oftwo single-mismatched sgRNAs is shown in white and labeled with “Com”.Fluorescence results represent average and SEM of three biologicalreplicates.

FIG. 51A-51C is related to FIG. 43F and depicts the combinatorialsilencing effects of using two sgRNAs to regulate a single gene. In allcases, non-overlapping sgRNAs showed augmentative silencing effects, andoverlapping sgRNAs showed suppressive effects. The combinatorial effectwas independent of whether the sgRNA was targeting the template ornon-template DNA strands. Fluorescence results represent average and SEMof three biological replicates.

Interrogating an Endogenous Regulatory Network Using CRISPRi GeneKnockdown

The CRISPRi system was next used as a gene knockdown platform tointerrogate endogenous gene networks. Previous methods to interrogatemicrobial gene networks have mostly relied on laborious and costlygenomic engineering and knockout procedures. By contrast, gene knockdownwith CRISPRi requires only the design and synthesis of a small sgRNAbearing a 20-bp complementary region to the desired genes. Todemonstrate this, CRISPRi was used to create E. coli knockdown strainsby designing sgRNAs to systematically perturb genes that were part ofthe well-characterized E. coli lactose regulatory pathway (FIG. 44A).β-galactosidase assays were performed to measure LacZ expression fromthe knockdown strains, with and without Isopropylβ-D-1-thiogalactopyranoside (IPTG), a chemical that inhibits the lacrepressor (LacI). In wild-type cells, addition of IPTG induced LacZexpression. The results showed that a lacZ-specific sgRNA could stronglyrepress LacZ expression (FIG. 44B). Conversely, an sgRNA targeting thelacI gene led to activation of LacZ expression, even in the absence ofIPTG, as would be expected for silencing a direct repressor of LacZexpression.

It is known that cAMP-CRP is an essential activator of LacZ expressionby binding to a cis regulatory site upstream of the promoter (A site).Consistently, the sgRNA that was targeted to the crp gene or to the Asite in the LacZ promoter led to repression, demonstrating a means tolink a regulator to its cis-regulatory sequence using CRISPRiexperiments. Targeting the adenylate cylase gene (cya), which isnecessary to produce the cAMP that makes CRP more effective at the LacZpromoter, only led to partial repression. Addition of 1 mM cAMP to thegrowth media complemented the effects for cya knockdown but not for crpknockdown, suggesting that cya is an indirect regulator of LacZ.Furthermore, targeting the LacI cis-regulatory site (O site) with ansgRNA led to inhibition, presumably because Cas9 complex binding at thissite sterically blocks RNA polymerase, mimicking the behavior of theLacI transcription repressor. Targeting the known RNAP binding site (Psite) also blocked expression. In summary, these studies demonstratethat the CRISPRi-based gene knockdown method provides a rapid andeffective approach for interrogating the regulatory functions(activating or repressing) of genes and cis elements in a complexregulatory network (FIG. 44C).

FIG. 44A-44C demonstrates functional profiling of a complex regulatorynetwork using CRISPRi gene knockdown. (FIG. 44A) sgRNAs were designedand used to knock down genes (cya, crp, lacI, lacZ, lacY, lacA) in thelac regulatory pathway or block transcriptional operator sites (A/P/O).LacI is a repressor of the lacZYA operon by binding to a transcriptionoperator site (O site). The lacZ gene encodes an enzyme that catalyzeslactose into glucose. A few trans-acting host genes such as cya and crpare involved in the activation of the lacZYA system. The cAMP-CRPcomplex binds to a transcription operator site (A site) and recruits RNApolymerase binding to the P site, which initiates transcription oflacZYA. IPTG, a chemical that inhibits LacI function, induces LacZexpression. (FIG. 44B) β-galactosidase assay of the knockdown strainswithout (white) and with (grey) IPTG. Control shows that the wild-typecells without CRISPRi perturbation could be induced by addition of IPTG.The sgRNA that targets LacZ strongly repressed LacZ expression, even inthe presence of IPTG. When LacI was targeted, LacZ expression was high,even without IPTG. Targeting cya and crp genes led to decreased LacZexpression level in the presence of IPTG. Presence of 1 mM cAMP rescuedcya knockdown but not crp knockdown. Blocking the transcription operatorsites resulted in LacZ repression, suggesting that these are importantcis-acting regulatory sites for LacZ. Upon perturbation, decreased (downarrows) and increased (up arrows) expression of LacZ is indicated. Errorbars represent SEM from three biological replicates. (FIG. 44C) Theknockdown experiments allowed us to profile the roles of regulators inthe lac regulatory circuit. The data is shown on a 2-D graph, withx-axis showing LacZ activity without IPTG and y-axis showing itsactivity with IPTG. The spreading of ovals along each axis shows thestandard deviations. The 0-galactosidase assay results represent averageand SEM of three biological replicates. For RNA-seq data on LacI andLacZ targeting, see also FIG. 49A-49C.

CRISPRi can Knock Down Targeted Gene Expression in Human Cells

To test the generality of the CRISPRi approach for using the dCas9-sgRNAcomplex to repress transcription, the system was tested in HEK293mammalian cells. The dCas9 protein was codon optimized, fused to threecopies of a nuclear localization sequence (NLS), and expressed from aMurine Stem Cell Virus (MSCV) retroviral vector. The same sgRNA designshown in FIG. 40B was used to express from the RNA polymerase III U6promoter. A reporter HEK293 cell line expressing EGFP under the SV40promoter was created by viral infection. Using an sgRNA (eNT2) thattargeted the non-template DNA strand of the EGFP coding region, amoderate but reproducible knockdown of gene expression was observed (46%repression, FIG. 45A). The repression was dependent on both the dCas9protein and sgRNA, implying that repression was due to the dCas9-sgRNAcomplex and RNA-guided targeting. The same sgRNA exhibited betterrepression on the same gene when transiently expressed from a plasmid(63% repression, FIG. 52). Consistent with the bacterial system, onlysgRNAs targeted to the non-template strand exhibited repression. Factorssuch as the distance from the transcription start and the localchromatin state may be critical parameters determining repressionefficiency (FIG. 52). Optimization of dCas9 and sgRNA expression,stability, nuclear localization, and interaction will allow for furtherimprovement of CRISPRi efficiency in mammalian cells.

FIG. 45A-45B demonstrates that CRISPRi can repress gene expression inhuman cells. (FIG. 45A) A CRISPRi system in HEK293 cells. The SV40-EGFPexpression cassette was inserted into the genome via retroviralinfection. The dCas9 protein was codon-optimized and fused with threecopies of NLS sequence. The sgRNA was expressed from an RNA polymeraseIII U6 vector. Co-transfection of dCas9 and an sgRNA (eNT2) that targetsthe non-template strand of EGFP decreased fluorescence (˜46%) while theexpression of either dCas9 or sgRNA alone showe no effect. (FIG. 45B)The dCas9:sgRNA-mediated repression was dependent on the target loci.Seven sgRNAs were designed to target different regions of the EGFPcoding sequence on the template or non-template strand. Only eNT2 andeNT5 showed moderate repression. Fluorescence results from 7A and 7Brepresent average and error of two biological replicates.

FIG. 52 is related to FIG. 45A-45B and shows that sgRNA repression isdependent on the target loci and relatively distance from thetranscription start. The same sgRNA was used to repress the same EGFPgene with different promoters. Cas9/sgRNA complexes repressedtranscription from transiently transfected plasmid DNA. The level oftranscriptional repression was slightly better (63%) than that observedfor genomic genes, and the percentage of GFP-negative cells increased inthe presence of sgRNA. The target locus has different distance from thetranscription start. While SV40-EGFP showed repression, LTR-EGFP had noeffect. Fluorescence results represent average and error of twobiological replicates.

CRISPRi Efficiently and Selectively Represses Transcription of TargetGenes

The CRISPRi system is a relatively simple platform for targeted generegulation. CRISPRi does not rely on the presence of complex hostfactors, but instead only requires the dCas9 protein and guide RNAs, andthus is flexible and highly designable. The system can efficientlysilence genes in bacteria. The silencing is very efficient, as nooff-target effects were detected. Furthermore, the efficiency of theknockdown can be tuned by changing the target loci and the degree ofbasepairing between the sgRNA and the target gene. This will make itpossible to create allelic series of hypomorphs—a feature that isespecially useful for the study of essential genes. The system functionsby directly blocking transcription, in a manner that can be easilyprogrammed by designing sgRNAs. Mechanistically, this is distinct fromRNAi-based silencing, which requires the destruction of alreadytranscribed mRNAs.

In addition, these dCas9:sgRNA complexes can also modulate transcriptionby targeting key cis-acting motifs within any promoter, stericallyblocking the association of their cognate trans-acting transcriptionfactors. Thus, in addition to its use as a gene knockdown tool, CRISPRicould be used for functional mapping of promoters and other genomicregulatory modules.

CRISPRi is Amenable to Genome-Scale Analysis and Regulation

The CRISPRi method is based on the use of DNA-targeting RNAs, and onlythe DNA targeting segment needs to be designed for specific genetargets. With the advances of large-scale DNA oligonucleotide synthesistechnology, generating large sets of oligonucleotides that containunique 20-bp regions for genome targeting is fast and inexpensive. Theseoligonucleotide libraries could allow us to target large numbers ofindividual genes to infer gene function or to target gene pairs to mapgenetic interactions. Furthermore, CRISPRi could be used tosimultaneously modulate the expression of large sets of genes, as thesmall size of sgRNAs allows one to concatenate multiple elements intothe same expression vector.

CRISPRi Provides New Tools for Manipulating Microbial Genomes

Because the CRISPRi platform is compact and self-contained, it can beadapted for different organisms. CRISPRi is a powerful tool for studyingnon-model organisms for which genetic engineering methods are not welldeveloped, including pathogens or industrially useful organisms. Unlikemost eukaryotes, most bacteria lack the RNAi machinery. As aconsequence, regulation of endogenous genes using designed syntheticRNAs is currently limited. CRISPRi could provide an RNAi-like method forgene perturbation in microbes.

CRISPRi as a Platform for Engineering Transcriptional RegulatoryNetworks

The CRISPRi can be utilized as a flexible framework for engineeringtranscriptional regulatory networks. The CRISPRi platform, because it isessentially a RNA-guided DNA-binding complex, also provides a flexiblescaffold for directing diverse regulatory machinery to specific sites inthe genome. Beyond simply blocking transcription of target genes, it ispossible to couple the dCas9 protein with numerous regulatory domains tomodulate different biological processes and to generate differentfunctional outcomes (e.g transcriptional activation, chromatinmodifications).

In the CRISPRi system, it is possible to link multiple sgRNAs intotranscriptional circuits in which one upstream sgRNA controls theexpression of a different downstream sgRNA. As RNA molecules inmicroorganisms tend to be short-lived, we suspect that the geneticprograms regulated by sgRNAs might show rapid kinetics distinct fromcircuits that involve slow processes such as protein expression anddegradation. In summary, the CRISPRi system is a general geneticprogramming platform suitable for a variety of biomedical research andclinical applications including genome-scale functional profiling,microbial metabolic engineering, and cell reprogramming.

Example 5: A Chimeric Site-Directed Polypeptide can be Used to Modulate(Activate or Repress) Transcription in Human Cells

We have demonstrated that in human cells, a fusion protein comprising acatalytically inactive Cas9 and an activator domain or a repressordomain can increase or decrease transcription from a target DNA,respectively.

FIG. 55A-55D. We fused the humanized catalytically inactive Cas9 with atranscription activator domain VP64. (FIG. 55A) To test the efficiencyfor gene activation using this system, we inserted a GAL4 UAS induciblepromoter that controls a GFP into the HEK293 (human tissue culturecells) genome. (FIG. 55B) The GAL4 UAS promoter can be induced in thepresence of yeast-derived protein GAL4. The dCas9-VP64 fusion caneffectively activate GAL4 UAS by 20-fold in the presence of a cognateguide RNA that binds to the GAL4 UAS region. (FIG. 55C) Microscopicimages for dCas9-VP64 activation. (FIG. 55D) Flow cytometry data fordCas9-VP64 activation.

FIG. 56 We fused the humanized catalytically inactive Cas9 with atranscription repressor domain KRAB. (Top) We designed 10 guide RNAsthat target a well-characterized promoter SV40 early promoter and oneguide RNA that targets the EGFP coding region. (Bottom) Using anon-chimeric dCas9, we observed 2-3 fold repression for gRNAs of P9 andNT2. This efficiency was greatly improved using the dCas9-KRAB fusion.For example, with dCas9-KRAB fusion, P9 and NT2 showed 20-fold and15-fold repression, respectively. In addition P1-P6 showed a significantreduction in expression when the fusion protein was used, but limitedrepression when a non-chimeric dCas9 was used.

Example 6: Cas9 can Use Artificial Guide RNAs, not Existing in Nature,to Perform Target DNA Cleavage

An artificial crRNA and an artificial tracrRNA were designed based onthe protein-binding segment of naturally occurring transcripts of S.pyogenes crRNA and tracrRNAs, modified to mimic the asymmetric bulgewithin natural S. pyogenes crRNA:tracrRNA duplex (see the bulge in theprotein-binding domain of both the artificial (top) and natural (bottom)RNA molecules depicted in FIG. 57A). The artificial tracrRNA sequenceshares less than 50% identity with the natural tracrRNA. The predictedsecondary structure of the crRNA:tracrRNA protein-binding duplex is thesame for both RNA pairs, but the predicted structure of the rest of theRNAs is much different.

FIG. 57A-57B demonstrates that artificial sequences that share verylittle (roughly 50% identity) with naturally occurring a tracrRNAs andcrRNAs can function with Cas9 to cleave target DNA as long as thestructure of the protein-binding domain of the DNA-targeting RNA isconserved. (FIG. 57A) Co-folding of S. pyogenes tracrRNA and crRNA andartificial tracrRNA and crRNA. (FIG. 57B) Combinations of S. pyogenesCas9 and tracrRNA:crRNA orthologs were used to perform plasmid DNAcleavage assays. Spy—S. pyogenes, Lin—L. innocua, Nme—N. meningitidis,Pmu—P. multocida. S. pyogenes Cas9 can be guided by some, but not alltracrRNA:crRNA orthologs naturally occuring in selected bacterialspecies. Notably, S. pyogenes Cas9 can be guided by the artificialtracrRNA:crRNA pair, which was designed based on the structure of theprotein-binding segment of the naturally occurring DNA-targeting RNAusing sequence completely unrelated to the CRISPR system.

The artificial “tracrRNA” (activator RNA) used was (SEQ ID NO: 1347)5′-GUUUUCCCUUUUCAAAGAAAUCUCCUGGGCACCUAUCUUCUUAGGUGCCCUCCCUUGUUUAAACCUGACCAGUUAACCGGCUGGUUAGGUUUUU-3′.The artificial “crRNA” (targeter RNA) used was: (SEQ ID NO: 1348)5′-GAGAUUUAUGAAAAGGGAAAAC-3′.

Example 7: Generation of Non-Human Transgenic Organisms

A transgenic mouse expressing Cas9 (either unmodified, modified to havereduced enzymatic activity, modified as a fusion protein for any of thepurposes outline above) is generated using a convenient method known toone of ordinary skill in the art (e.g., (i) gene knock-in at a targetedlocus (e.g., ROSA 26) of a mouse embryonic stem cell (ES cell) followedby blastocyst injection and the generation of chimeric mice; (ii)injection of a randomly integrating transgene into the pronucleus of afertilized mouse oocyte followed by ovum implantation into apseudopregnant female; etc.). The Cas9 protein is under the control of apromoter that expresses at least in embryonic stem cells, and may beadditionally under temporal or tissue-specific control (e.g., druginducible, controlled by a Cre/Lox based promoter system, etc.). Once aline of transgenic Cas9 expressing mice is generated, embryonic stemcells are isolated and cultured and in some cases ES cells are frozenfor future use. Because the isolated ES cells express Cas9 (and in somecases the expression is under temporal control (e.g., drug inducible),new knock-out or knock-in cells (and therefore mice) are rapidlygenerated at any desired locus in the genome by introducing anappropriately designed DNA-targeting RNA that targets the Cas9 to aparticular locus of choice. Such a system, and many variations thereof,is used to generate new genetically modified organisms at any locus ofchoice. When modified Cas9 is used to modulate transcription and/ormodify DNA and/or modify polypeptides associated with DNA, the ES cellsthemselves (or any differentiated cells derived from the ES cells (e.g.,an entire mouse, a differentiated cell line, etc.) are used to study toproperties of any gene of choice (or any expression product of choice,or any genomic locus of choice) simply by introducing an appropriateDNA-targeting RNA into a desired Cas9 expressing cell.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A composition comprising a single moleculeDNA-targeting RNA, or a nucleic acid encoding said single moleculeDNA-targeting RNA, wherein the single molecule DNA-targeting RNAcomprises, in 5′ to 3′ order: (i) a targeter-RNA comprising (a) aDNA-targeting sequence, which is a first nucleotide sequence that iscomplementary to a target sequence of a target DNA, and (b) a secondnucleotide sequence that hybridizes with an activator-RNA, wherein thefirst and second nucleotide sequences are heterologous to one another;and (ii) the activator-RNA, which hybridizes with the targeter-RNA toform a double-stranded duplex, wherein (i) and (ii) are covalentlylinked by intervening nucleotides, and wherein the single moleculeDNA-targeting RNA is capable of forming a complex with a Cas9 proteinand guiding the complex to the target sequence to cleave the target DNA.2. The composition of claim 1, wherein the DNA-targeting sequence is 18to 30 nucleotides long.
 3. The composition of claim 1, wherein saidintervening nucleotides are 3-5 nucleotides.
 4. The composition of claim1, wherein the activator-RNA comprises the 67 nucleotide tracrRNAsequence (SEQ ID NO: 432)UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUUor the 26 nucleotide tracrRNA sequence (SEQ ID NO: 441)UAGCAAGUUAAAAUAAGGCUAGUCCG.


5. The composition of claim 4, wherein the targeter-RNA comprises the 12nucleotide (nt) crRNA sequence GUUUUAGAGCUA (SEQ ID NO: 679).
 6. Thecomposition of claim 1, comprising said single molecule DNA-targetingRNA, wherein the single molecule DNA-targeting RNA comprises one or morephosphorothioates, one or more inverted polarity linkages, one or moreabasic nucleoside linkages, one or more locked nucleic acids (LNAs), oneor more 2′-substituted sugar moieties, one or more 2′-O-methoxyethylmodified sugar moieties, one or more 2′-O-methyl modified sugarmoieties, one or more 2′-O-(2-methoxyethyl) modified sugar moieties, oneor more 2′-fluoro modified sugar moieties, one or more2′-dimethylaminooxyethoxy modified sugar moieties, one or more2′-dimethylaminoethoxyethoxy modified sugar moieties, one or morepeptide nucleic acids (PNAs), one or more morpholino nucleic acids, oneor more cyclohexenyl nucleic acids (CeNAs), or any combination thereof.7. The composition of claim 1, further comprising the Cas9 protein or anucleic acid encoding the Cas9 protein.
 8. The composition of claim 7,comprising a protein-RNA complex comprising the Cas9 protein and thesingle molecule DNA-targeting RNA.
 9. A composition comprising a singlemolecule DNA-targeting RNA, or a nucleic acid encoding said singlemolecule DNA-targeting RNA, wherein the single molecule DNA-targetingRNA comprises, in 5′ to 3′ order: (i) a targeter-RNA comprising (a) aDNA-targeting sequence, which is a first nucleotide sequence that iscomplementary to a target sequence of a target DNA, and (b) a secondnucleotide sequence that hybridizes with an activator-RNA; and (ii) theactivator-RNA, which hybridizes with the targeter-RNA to form adouble-stranded duplex, wherein (i) and (ii) are covalently linked byintervening nucleotides, wherein the single molecule DNA-targeting RNAis capable of forming a complex with a Cas9 protein and guiding thecomplex to the target sequence to cleave the target DNA, and wherein:(A) the composition is lyophilized; or (B) the composition comprises anuclease inhibitor, a buffering agent, a detergent, a polyamine, anadjuvant, a wetting agent, a stabilizing agent, an antioxidant, acomplexing agent, or any combination thereof.
 10. The composition ofclaim 9, wherein the composition comprises the nuclease inhibitor. 11.The composition of claim 9, wherein the composition is lyophilized. 12.The composition of claim 9, wherein the Cas9 protein is a Streptococcuspyogenes Cas9.
 13. The composition of claim 9, further comprising theCas9 protein or a nucleic encoding the Cas9 protein.
 14. The compositionof claim 13, wherein the Cas9 protein comprises one or more mutations ina RuvC domain and/or an HNH domain.
 15. The composition of claim 9,wherein the DNA-targeting sequence is 18 to 30 nucleotides long.
 16. Thecomposition of claim 9, wherein the activator-RNA comprises the 67nucleotide tracrRNA sequence (SEQ ID NO: 432)UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUUor the 26 nucleotide tracrRNA sequence (SEQ ID NO: 441)UAGCAAGUUAAAAUAAGGCUAGUCCG.


17. The composition of claim 16, wherein the targeter-RNA comprises the12 nucleotide (nt) crRNA sequence GUUUUAGAGCUA (SEQ ID NO: 679).
 18. Acomposition comprising a single molecule DNA-targeting RNA, or a nucleicacid encoding said single molecule DNA-targeting RNA, wherein the singlemolecule DNA-targeting RNA comprises, in 5′ to 3′ order: (i) atargeter-RNA comprising (a) a DNA-targeting sequence, which is a firstnucleotide sequence that is complementary to a target sequence of atarget DNA, and (b) a second nucleotide sequence that hybridizes with anactivator-RNA; and (ii) the activator-RNA, which hybridizes with thetargeter-RNA to form a double-stranded duplex, wherein (i) and (ii) arecovalently linked by 3-20 intervening nucleotides, and wherein thesingle molecule DNA-targeting RNA is capable of forming a complex with aCas9 protein and guiding the complex to the target sequence to cleavethe target DNA.
 19. The composition of claim 18, wherein theDNA-targeting sequence is 18 to 30 nucleotides long.
 20. The compositionof claim 18, wherein the activator-RNA comprises the 67 nucleotidetracrRNA sequence (SEQ ID NO: 432)UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUUor the 26 nucleotide tracrRNA sequence (SEQ ID NO: 441)UAGCAAGUUAAAAUAAGGCUAGUCCG.


21. The composition of claim 20, wherein the targeter-RNA comprises the12 nucleotide (nt) crRNA sequence GUUUUAGAGCUA (SEQ ID NO: 679).
 22. Thecomposition of claim 18, wherein the composition comprises said Cas9protein or a nucleic encoding the Cas9 protein.
 23. The composition ofclaim 22, wherein the Cas9 protein is a Streptococcus pyogenes Cas9. 24.The composition of claim 22, comprising a protein-RNA complex comprisingthe Cas9 protein and the single molecule DNA-targeting RNA.
 25. Acomposition comprising: a nucleic acid that comprises a nucleotidesequence encoding a single molecule DNA-targeting RNA, wherein thesingle molecule DNA-targeting RNA comprises, in 5′ to 3′ order: (i) atargeter-RNA comprising (a) a DNA-targeting sequence, which is a firstnucleotide sequence that is complementary to a target sequence of atarget DNA, and (b) a second nucleotide sequence that hybridizes with anactivator-RNA; and (ii) the activator-RNA, which hybridizes with thetargeter-RNA to form a double-stranded duplex, wherein (i) and (ii) arecovalently linked by intervening nucleotides, and wherein the singlemolecule DNA-targeting RNA is capable of forming a complex with a Cas9protein and guiding the complex to the target sequence to cleave thetarget DNA, wherein the nucleotide sequence encoding a single moleculeDNA-targeting RNA is operably linked to a heterologous promoter.
 26. Thecomposition of claim 25, wherein said nucleic acid is a plasmid, acosmid, a minicircle, a phage, or a viral vector.
 27. The composition ofclaim 25, wherein said nucleic acid is a retroviral, lentiviral,adenoviral, adeno-associated, or herpes simplex virus vector.
 28. Thecomposition of claim 25, wherein the heterologous promoter is aninducible promoter.
 29. The composition of claim 25, wherein theactivator-RNA comprises the 67 nucleotide tracrRNA sequence(SEQ ID NO: 432) UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU or the 26 nucleotide tracrRNA sequence (SEQ ID NO: 441)UAGCAAGUUAAAAUAAGGCUAGUCCG.


30. The composition of claim 29, wherein the targeter-RNA comprises the12 nucleotide (nt) crRNA sequence GUUUUAGAGCUA (SEQ ID NO: 679).